HUT66828A - Single-stranded circular oligonucleotides - Google Patents

Abstract

The present invention provides single-stranded circular oligonucleotides each with a parallel binding (P) domain and an anti-parallel binding (AP) domain separated from each other by loop domains. Each P and AP domain has sufficient complementarity to bind to one strand of a defined nucleic acid target wherein the P domain binds in a parallel manner to the target and the AP domain binds in an anti-parallel manner to the target. Moreover, the present single-stranded circular oligonucleotides can bind to both single-stranded and double-stranded target nucleic acids. The present invention also provides methods of using these oligonucleotides as well as pharmaceutical compositions containing these oligonucleotides.

Description

Institute provided it. The United States Government has certain rights in the invention.

The present invention provides single-stranded circular oligonucleotides that are capable of binding to a target DNA or a target RNA, thereby controlling DNA replication, RNA transcription, protein translation, and other processes involving nucleic acid templates. Further, circular oligonucleotides may be labeled for use as probes for the detection or isolation of a target nucleic acid. Circular oligonucleotides can displace a strand of a duplex nucleic acid without previously denaturing the duplex. Furthermore, circular oligonucleotides are resistant to exonucleases and bind to the target with greater selectivity and affinity than linear oligonucleotides.

The background of the invention is described below

An oligonucleotide binds to a target nucleic acid by the formation of hydrogen bonds between the target and the base of the oligonucleotide. Generic B-DNA has conventional adenine-thymine (A-T) and guanine-cytosine (G-C) Watson and Crick base pairs, with 2 and 3 hydrogen bonds, respectively. Conventional hybridization techniques rely on the ability of sequence-specific DNA or RNA probes to bind to a target nucleic acid via Watson-Crick hydrogen bonds. However, other types of hydrogen bonding modes are known in which some atoms of a base that are not involved in the Watson-Crick base formation can form hydrogen bonds with other nucleotides. For example, thymine (T) may bind to an A-T Watson-Crick base pair associated with adenine * · ·

3 through hydrogen bonds to form an A-AT base triad. Hoogsteen (1959, Acta Crystallography 12: 822) first described the alternative hydrogen bonds present in the T-AT and C-GC base triads. Later, G-TA base triads in which guanine can form a hydrogen bond with a central thymine were observed (Griffin et al., 1989, Science 245: 967-971). If an oligonucleotide is capable of binding to one target via both Watson-Crick and alternative hydrogen bonds, an extremely stable complex can be formed which has many in vivo and in vitro applications. However, no oligonucleotide having the necessary structural properties necessary to achieve binding to Watson-Crick and alternative hydrogen bonds has yet been described.

Oligonucleotides that did not bind to WatsonCrick's hydrogen bonds in vitro have been observed. For example, Cooney et al., (1988, Science 241: 456) describe a 27-base single-stranded oligonucleotide that binds to a double-stranded nucleic acid via non-WatsonCrick hydrogen bonds. However, the triple-stranded complexes of this type are not very stable since they are bound to the oligonucleotide target only by less stable alternative hydrogen bonds, e.g. without a single Watson-Crick bond.

Oligonucleotides are used for many purposes. For example, oligonucleotides can be used as probes for target nucleic acids immobilized on a filter or membrane or present in tissue. Sambrook et al (1989, Molecular Cloning:

A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, NY) provide a detailed description of hybridization techniques.

Furthermore, there is great interest nowadays in the development of oligonucleotides as regulators of biological functions of cellular nucleic acids. This interest stems from the observation of naturally occurring complementary or antisense RNA that is used by some cells to regulate protein expression. However, the development of oligonucleotides for the in vivo regulation of biological processes is hampered by a number of longstanding problems, including low binding stability of the linear oligonucleotide and nuclease sensitivity.

For example, transcription of the human c-mvc gene was inhibited in a cell-free, in vivo experimental system by a 27-base linear oligonucleotide designed to bind to the c-mvc promoter. Inhibition was observed only when a carefully controlled in vitro assay system was used, which used lower than physiological temperature and removed or inactivated many cellular enzymes. These conditions were required because linear oligonucleotides bind with low affinity and are highly sensitive to enzymes that break down linear fragments of DNA (Cooney et al.). Coupling of a pre-mRNA transcript, essential for replication of the Herpes Simplex virus, was also inhibited by a linear oligonucleotide that was complementary to an acceptor binding site. In this case, a methylphosphonate bond was used in the linear oligonucleotide to enhance nuclease resistance. Addition of this chemically modified oligonucleotide to the culture medium caused a decrease in protein synthesis and in the proliferation of uninfected cells, probably due to toxicity problems at high concentrations (Smith et al., 1986, Proc. Natl. Acad. Sci. 82: 2787-2791).

In another example, linear oligonucleotides were used to inhibit human immunodeficiency virus replication in cultured cells. Linear oligonucleotides close to, or complementary to, sites within the retroviral genome and complementary to certain linkage sites within the retroviral genome were most effective in blocking viral replication. However, large amounts of linear oligonucleotides were required in these experiments to achieve the effect, presumably because these linear oligonucleotides have low binding stability and are readily damaged by nucleases (Goodchild et al., 1988, Proc. Natl. Acad. Sci. USA 85). : 5507-5511).

Accordingly, oligonucleotides which are useful regulators of biological processes preferably have certain properties. First, the oligonucleotide must bind strongly enough to the complementary target nucleic acid to have the desired regulatory effect. Second, it is generally desirable that the oligonucleotide and its target be sequence specific. Third, the oligonucleotide has a sufficient half-life under in vivo conditions to allow the desired regulation in the cell. However, the oligonucleotide must be resistant to enzymes that cleave nucleic acids, e.g. the kinks. Fourth, the oligonucleotide must be capable of binding to single-stranded and double-stranded targets.

While linear oligonucleotides can satisfy the sequence specificity requirements, linear oligonucleotides are sensitive to nucleases and generally require chemical modification to increase their biological half-lives. Such modifications increase the cost of oligonucleotide production and may also cause toxicity problems. In addition, linear oligonucleotides bind to form a double-stranded complex, which is present in cellular nucleic acids. Consequently, cellular enzymes can readily modify and dissociate the target linear oligonucleotide in the double-stranded complex. The low binding strength and nuclease sensitivity of the linear oligonucleotides may thus necessitate the use of high concentrations of the oligonucleotide, thus rendering such application toxic or costly. Furthermore, while linear oligonucleotides can be linked to a double-stranded target by alternative hydrogen bonds (e.g., Hoogstein's bond), linear oligonucleotides cannot readily dissociate the double-stranded target to replace one strand and thus provide a more stable Watson-Crick binding pattern. created.

In addition, increased binding strength enhances the efficiency of the regulatory oligonucleotide. Therefore, a higher binding affinity oligonucleotide can be used at a lower dose. A lower dose reduces costs and reduces the likelihood of toxicity.

Accordingly, the present invention provides single-stranded circular oligonucleotides which, due to the nature of the circularity of the oligonucleotide and the domains present on the oligonucleotide, are nuclease resistant and bind to their target nucleic acids with high affinity and high selectivity. Further, the circular oligonucleotides of the invention can dissociate and bind to the double-stranded target without having previously been denatured.

Some types of single-stranded DNA or RNA circuits are known.

For example, some naturally occurring viral and bacteriophage genomes have the structure of a single-stranded circular nucleic acid. Single-stranded DNA circuits are described by Erié et al. (1987, Biochemistry. 26: 7150-7159 and 1989,

Biochemistry 28: 268-273). However, none of these circular molecules have been designed to bind to the target nucleic acid. However, the present invention presents an innovation characterized by a fundamental improvement unlike prior art in this field because of the high specificity of circular oligonucleotides with low or non-existent toxicity and greater resistance to nucleases than linear oligonucleotides, whereas the single-stranded or double-stranded target its binding to nucleic acids is much stronger than that of conventional linear oligonucleotides.

• · «» ·· ···

The following is a summary of the invention

The present invention provides a single-stranded circular oligonucleotide having at least one parallel-linked (P) domain and at least one non-parallel-linked (AP) domain and having a loop domain between each binding domain to generate a circular oligonucleotide. Each of the P and corresponding AP domains has sufficient complementarity to detectably bind to a strand of a particular nucleic acid target by binding parallel to the target P domain and the AP domain to an anti-parallel binding site. Sufficient complementarity means that there is a sufficient number of base pairs between the P and / or AP domains of the target nucleic acid and the circular oligonucleotide, e.g. to achieve a detectable bond.

Another embodiment of the invention provides single-stranded circular oligonucleotides that are derived from a reporter molecule, are derived from a drug or other pharmaceutical material to provide probe for the target nucleic acid, or are derived from other substances that are capable of cleavage. modified from the target nucleic acid or from substances that promote cellular uptake or binding of the target oligonucleotide.

In a further embodiment of the invention, single-stranded circular oligonucleotides are provided which are linked to a solid support to isolate the nucleic acid complementary to the oligonucleotide.

In yet another embodiment of the present invention, there is provided a split kit for detecting or diagnosing a target nucleic acid, including at least a first storage portion that provides any of the circular oligonucleotides.

In a further embodiment of the invention, there is provided a method of detecting a target nucleic acid comprising contacting a single-stranded circular oligonucleotide with a sample comprising the target nucleic acid for a period and under conditions suitable for the formation of the oligonucleotide-target complex and complex. detection. This detection method may be fluorescent energy transfer.

In yet another embodiment of the invention, there is provided a method of regulating DNA, RNA and protein biosynthesis. This method involves combining at least one circular oligonucleotide with a DNA, RNA, or protein nucleic acid template under conditions sufficient to achieve binding to the target sequence in the oligonucleotide template, followed by binding to the oligonucleotide, and access to the template is therefore blocked. , RNA or protein biosynthesis is regulated.

In a further embodiment of the invention, pharmaceutical compositions are provided for controlling the biosynthesis of at least one of the circular oligonucleotides that control the biosynthesis of a nucleic acid or protein, and a pharmaceutically acceptable carrier.

In a further embodiment of the invention, there is provided a method of producing a single-stranded circular oligonucleotide which comprises binding to a terminal end-linked oligonucleotide of a linear lifetime and recovering the circular oligonucleotide product.

In yet another embodiment of the invention, there is provided a method of strand displacement in a double-stranded nucleic acid target by contacting the target with any of the circular oligonucleotides for such time and under conditions sufficient to denature the target and bind the circular oligonucleotide.

The following is a brief description of the drawings of the invention

IA. Fig. 3A shows the binding pattern of AT and GC base pairs of Watson-Crick (anti-parallel domain). 1B. Figures 3A and 4B show the base triads of T-AT, C + GC and G-TA which may be formed between P, target and AP nucleotides.

Figure 2 schematically illustrates the circularization reaction required for the synthesis of single-stranded circular oligonucleotides. A linear age oligonucleotide binds to an oligonucleotide having the same sequence as the target, e.g. an end-linked oligonucleotide to form a complex of age. After ligation, the circularized oligonucleotides were separated from the end-linked oligonucleotides.

Fig. 3 from linear precursors to circular oligo ♦ ♦ ·

shows sequences up to nucleotides, e.g. precursors (1-3 with SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7), goals (4-5 with SEQ IDs) NO: 8 and SEQ ID NO: 9), circular oligonucleotides (SEQ ID NOs: 5-7 and 14) and linear oligonucleotides (SEQ ID NOs: 5-7 and 14) and linear oligonucleotides (SEQ ID NOs: 5-7 and 14); 9-12 and 14 with SEQ ID NOs: 10-13 and 15), which are described in the Examples.

Figure 4 shows a linear lifetime structure complexed with an end-linked oligonucleotide before ligation.

Figure 5 shows the effect of pH on circular oligonucleotide: target complex formation as measured by Tm. The black circles show the stability of a 6: 4 complex at various pH values, while the black circles show the stability of the 7: 5 complexes. THE

The sequences of circular oligonucleotides 6 and 7 and the targets 4 and 5 are shown in Figure 3.

6A. Fig. 3A shows the effect of loop size on complex formation by comparing binding to two targets: a single (dA) ₁₂ target (squares) and a 36 nucleotide oligonucleotide target (s). 6B. Figure 3B shows the effect of target and binding domain lengths on complex formation.

Figure 7 shows the formation of a complex between a circular oligonucleotide and a target where the P and AP binding domains are staggered at the target.

• «« · · * • «· · · · · · · · · · · · · · ·

Fig. 8 is a graph showing one of the strands of a fluorescent labeled double-stranded target (SEQ ID NO: 11) with either a linear oligonucleotide having SEQ ID NO: 8 (dotted line) or a circular oligonucleotide having SEQ ID NO: 5 (solid line). ). Standard substitution was viewed by measuring the increase in fluorescence fluorescence intensity (Y-axis) versus time (X-axis).

Figure 9 uses the duplex (SEQ ID NO: 5) depicts the various concentrations C _{o and} b for the observed pseudo-first order rate constants of points. The uncertainty of the speed constants is not greater than 10%. The drawn curve is a right-angled hyperbola, created with the best coincidence. Double reciprocal editing of data eg. [circular oligonucleotide] ^-1 versus ( ^K _o b _s ) ^-1 linear with a slope of 8.95 x 1ο ^- θ sec.M ^-1 and a 39.8 second y-arrest.

The following is a detailed description of the invention.

The present invention relates to single-stranded circular oligonucleotides, e.g. circles that bind to nucleic acid targets with higher affinity and selectivity than their corresponding linear oligonucleotides. Furthermore, since these circles are capable of opening and binding to two strands of a double-stranded nucleic acid, both single-stranded and double-stranded nucleic acids can serve as targets for binding to circular oligonucleotides.

Furthermore, since the binding of these circuits to single-stranded and double-stranded targets is strong and selective, they provide a wide range of applications, including methods for controlling biological processes such as DNA replication, RNA transcription, RNA splicing and utilization, protein translation and the like. Similarly, the ability of these circuits to dissociate double-stranded nucleic acids and selectively and strongly bind to the targeted nucleic acid makes them ideal as diagnostic probes or markers to localize, e.g. specific sites on the chromosome or other DNA or RNA molecules. Furthermore, these circles are useful for isolating complementary nucleic acids or for sequence-specific delivery of drugs or other molecules to cells.

Preferably, the single-stranded circular oligonucleotides of the invention have at least one parallel-linked (P) domain and at least one anti-parallel-linked (AP) domain and have a loop domain between each binding domain, thereby forming a circular oligonucleotide. Further, each of the P and AP domains exhibits sufficient complementarity for binding to a single strand of a specific nucleic acid target by binding the P domain in a parallel manner to the target and the AP domain binding in an anti-parallel manner.

The schematic representation presented below illustrates the circular arrangement of a series of P and AP oligonucleotide domains relative to each other and linked to the target (T, as indicated below).

• · · «· •» ·· ♦ ··

- 14 ···

Ρ

------------> T <=== - AP

The arrows indicate the orientation of each strand from 5 'to 3', with the 5 'end at the tail portion of each domain and the 3' end at the tip of the arrows. However, as used herein, parallel binding of nucleic acids means that the 5 'to 3' orientation is the same for each strand or nucleotide in the complex. This type of binding is located between the target and the P domain. As used in the present invention, the anti-parallel binding of the nucleic acid means that the 5 'to 3' orientations of the two strands or nucleotides in the complex lie in opposite directions, e.g. the strands are located as they are in double helix DNA in a typical Watson-Crick base pair arrangement.

If more than one P or AP binding domain is present, such binding domains are separated from other P and AP domains by loop domains of sufficient length to allow binding to multiple targets. Furthermore, if a circle has multiple AP and P domains, the corresponding targets do not necessarily have to be on a nucleic acid strand. In addition, a circular oligonucleotide that binds to a particular target may be a loop domain of an AP or P domain for binding to a second target when the circular oligonucleotide is released from the first target.

In agreement with the present invention, the nucleotide sequence of the P and AP domains can be determined from a specific sequence of the target nucleic acid by reference to the following rules for base pairing. The target may be single-stranded or double-stranded and has been selected for its known functional and structural properties. For example, some preferred targets may be a coding region, an origin of replication, a reverse transcriptase binding site, a transcriptional regulatory element, an RNA splice site, or a ribosome binding site, among others. The target can also be selected on the basis of its ability to detect or isolate a DNA or RNA template. Preferred targets are purine rich e.g. in adenine and guanine.

The nucleotide sequence of the target DNA or RNA may be known in whole or in part. Once the target nucleotide sequence is fully known, the sequences of the P and AP domains are designed with the required degree of complementarity to produce binding as can be detected by known methods, e.g. by changes in light absorption or fluorescence. In some cases, the target sequence may be represented by a consensus sequence or may be only partially known. For example, circular oligonucleotides (circles) that bind to the entire class of a target represented by a consensus sequence may be provided such that the target is consensus. «+ + 9 9 9 + 9 9 9 «···» «·· ···

- We design the P and AP domains from 16 sequences. In this case, the targets, some of which will fit exactly into the consensus sequence, others will have some mismatched bases which, in turn, are not large enough to prevent binding. Similarly, once a portion of the target sequence is known, one of ordinary skill in the art may refer to the following base pairing rules for designing circuits that are linked to the target with greater affinity than a linear oligonucleotide, the sequence of which corresponds to the circular oligonucleotide.

Thus, the invention also encompasses circles having P and AP domains sufficiently complementary to bind to a nucleic acid target in which the positions of the P and AP domains are determined from a sufficient number of nucleotide positions in the target sequence, and the base pairing rules of the invention. The number of defined (i.e. known) positions is the number of positions required to provide sufficient complementarity for binding of the oligonucleotides of interest to their targets as detected by standard techniques, including changes in light absorption due to binding or melting.

The base pairing rule of the present invention allows the P domain to bind to the target by creating base pairs where both the P domain and the target nucleotides have the same 5 'to 3' orientation. Preferably, these rules are satisfactory to the extent necessary for the purpose of the circular oligonucleotide »···

- 17, e.g. complementarity is not required to be 100% until binding can be detected. However, the general rules for determining the sequence of the P domain are as follows:

if the base at one position in the target is a guanine or a guanine analog, then P has a cytosine or an equivalent analog thereof at the appropriate position;

if the base at the position in the target is an adenine or an adenine analog, then P has a thymine or a uracil or a suitable analogue thereof at the appropriate position;

if the base at one position in the target is a thymine or a thymine analog, then P has a cytosine or a guanine or a suitable analogue thereof at the corresponding position;

when the base at one position in the target is a cytosine or a cytosine analog, P has a cytosine, thymine or uracil, or a suitable analogue thereof at the corresponding position; and when the base at one position in the target is a uracil or a uracil analog, P has a cytosine, a guanine, thymine, or a uracil, or a suitable analogue thereof at the appropriate position.

The base pairing rule of the present invention assures the AP domain to bind to the target by creating base pairs where the AP domain and the target nucleotides are in opposite orientation. Preferably, these rules are sufficient to the extent necessary to achieve detectable binding of the circular oligonucleotide to its target, e.g. the complementarity may be less than 100%. However, the base mating rule may be adhered to only to the extent necessary to achieve a sufficient degree of complementarity to establish a detectable bond between the circular oligonucleotide and its target.

Thus, the general rules for determining the AP domain sequence are as follows:

if the base at the position in the target is a guanine or a guanine analog, then the AP has a cytosine or a uracil or a suitable analogue thereof at the appropriate position;

if the base at one position in the target is an adenine or an adenine analog, then the AP has a thymine or a uracil or a suitable analogue thereof at the appropriate position;

if the base at one position in the target is a thymine or a thymine analog, then the AP has an adenine or a suitable analogue thereof at the corresponding position;

if the base at one position in the target is a cytosine or a cytosine analog, then the AP has a guanine or a suitable analogue thereof at the appropriate position;

if the base at one position in the target is a uracil or a uracil analog, then the AP has an adenine or a guanine or a suitable analogue thereof at the appropriate position.

In a preferred embodiment, P, AP and «4 44« ·· *

4 «4 4 4 · ··· 4 ······················································································································································· ·

Table 1 summarizes which nucleotide is capable of generating anti-parallel base pairs or parallel base pairs with a specific target nucleotide.

Table 1

Target nucleotide Anti-parallel Paralell domain nucleotide domain nucleotide

G C or U C THE T or U T or U T THE C or G c G C, T or U u The cut G C, G, T or U

(a) or a suitable analog

Two complementary single-stranded nucleic acids form a stable double helix (duplex) form when the strands bind or hybridize to each other in a typical Watson-Crick fashion, e.g. anti-parallel GC and AT base pairs. The present invention provides stable duplex and stable triplex formation when the P and AP domains have sufficient complementarity with the target sequence, a stable link between the circular oligonucleotide and the target molecule. ·· ·· »to create a stutter. Stable binding occurs when an oligonucleotide is detectably bound to the target under the desired conditions.

Complementarity between nucleic acids is the degree to which the bases of one strand of a nucleic acid bind hydrogen bonds or base pairs to the bases of the second strand of nucleic acids. However, complementarity can sometimes be conveniently described as a percentage, e.g. as the ratio of nucleotides that form base pairs between the two strands either within a specific region or within the domains of the two strands. In the present invention, satisfactory complementarity means that there are sufficient base pairs between the P and / or AP domains of the target nucleic acid and circular oligonucleotide to achieve detectable binding. Further, the degree of complementarity between the P domain and the target and the AP domain and the target need not be the same. When expressed or measured in terms of the proportion of base pairs formed, the degree of complementarity can vary from 30-40% complementarity to 100% complementarity. In general, the total degree of complementarity between the P or AP domain and the target is preferably at least 50%. However, the complementarity of the P domain to the target may sometimes be less than the complementarity of the AP domain to the target, e.g. the P domain may show 30% complementarity to the target, while the AP domain may show significantly greater complementarity, e.g. 50% to 100% complementarity.

In addition, the complementarity of the circular oligonucleotide of interest and its proper targets is detectable.

The degree of £ · <* · ··· ♦ ·· depends on the circumstances under which the bond is formed. It is well known that inter-strand linkage of nucleic acids is e.g. hybridization depends on factors other than the degree of difference between the two sequences. Such factors include the region's GC content, temperature, ionic strength, the presence of formamide, and the type of oppositely charged ions present. The effect of these conditions on the bond is well known to those skilled in the art. In addition, conditions are often determined by the application modes. For example, when a circular oligonucleotide is used, in vivo formamide will not be present and the strength of the ions and the type and temperature of the oppositely charged ions will correspond to the physiological conditions. The binding conditions can be manipulated in vitro to optimize the applicability of the oligonucleotides. A comprehensive method of quantifying and qualifying binding conditions, which allows one of ordinary skill in the art to construct oligonucleotides suitable for use under the desired conditions, Beltz et al. , (1983, Methods Enzmol. 100: 266-285) and Sambrook et al.

please.

Thus, for the purposes of the present invention, one of ordinary skill in the art can readily design a nucleotide sequence for the P and AP domains of the circular oligonucleotides of interest, which exhibits sufficient complementarity to detectably bind to its target sequence. The term "binding" or "stable bond" is used to mean that the oligo "4" · · · · · · · · · · · · · · · · · · · · · · · ·· ···

- 22 nucleotides in sufficient amount to be detectable in the target or hybridized state. The purpose of binding is to detect the physical or functional properties of a circular oligonucleotide complex.

The binding between a target and an oligonucleotide can be detected by any means known to those skilled in the art, including both functional and physical binding assays. Functionally, the binding can be detected by determining whether the binding has a detectable effect on biosynthetic processes (e.g., DNA replication, RNA transcription, protein translation, etc.).

Physical detection methods for binding of complementary DNA or RNA strands are well known in the art and include methods such as DNase I or chemical footprinting gel run and affinity cleavage assays as well as light absorption detection methods. For example, a method widely used for reasons of simplicity and reliability involves monitoring the change in the light absorption of a solution containing the target nucleic acid and oligonucleotide at 220 and 300 nm as a result of the gradual increase in temperature. When the oligonucleotide is bound to its target, a sudden increase in absorption is observed at a typical temperature as the oligonucleotide and target dissociate or melt.

The bond between an oligonucleotide and the target nucleic acid is often characterized by the temperature at which the oligonucleotide is 50 · · ·

- 23% melt away from your target. This temperature is the melting temperature (T _m ). Higher T _{m means} a stronger or more stable complex than a complex with lower T _m . The stability of a duplex is enhanced by increasing the G: C content, since the G: C base pair has three hydrogen bonds, while the A: T base pair has only two. The circular oligonucleotides of the invention provide additional hydrogen bonds and thus much greater stability, since both binding domains, the P and AP domains, are available for single nucleic acid binding. Three Therefore, created by the circular oligonucleotide linked to the goal should be higher than T _m to melt like a linear oligonucleotide duplexes and set up by a goal.

Circular oligonucleotides bind to a nucleic acid target through hydrogen bonds that are formed between the nucleotides of the binding domain and the target. The AP domain can bind by forming Watson-Crick hydrogen bonds (Figure 1). The P domain is capable of binding to target nucleotides by the formation of non-Watson-Crick hydrogen bonds (e.g., Figure 1 and Table 1). When two nucleotides from different strands of DNA or RNA form hydrogen bonds according to the base pairing rules given herein, one base pair or one duplex is formed. When a nucleotide from the AP domain and a nucleotide from the P domain bind to the same target nucleotide, a base triad is formed.

Base base pairing with a complementary target strand of a nucleic acid is less thermodynamically advantageous than Watson-Crick base pairing; however, when both

parallel and antiparallel pairing modes are present in a single molecule very stable complexes can be formed. Thus, the two opposing domains of a circular oligomer form a complex having a central target, showing a triplex structure, or a triple helical complex linked by two looped ends of the circle. For example, this arrangement allows four hydrogen bonds to be formed when two thymines bind to one target adenine, and five hydrogen bonds to form when two cytosines are bound to one target guanine.

Furthermore, due to the binding properties of the P and AP domains, the circular oligonucleotides of the invention are more selective for a particular target than the corresponding linear oligonucleotides. At least two factors contribute to this enhanced selectivity. First, the circular oligonucleotides of the invention bind twice to the same central target strand. Thus, two domains are involved in selecting one target. Second, in a C + G-C triad, the protonization of cytosine is only successful if this triad is formed and the additional proton gives a positive charge to the triad. This positive charge can reduce the negative charge repulsion resulting from the juxtaposition of the three phosphodiester backbones.

Unlike linear oligonucleotides, circular oligonucleotides of the invention may displace one strand of a double-stranded target under conditions where the denaturation of the double-stranded target is not thermodynamically favorable. Linear oligonucleotides do not have the ability to displace a strand of a duplex. For example, the half-life of a double-stranded target in the presence of a complementary linear oligonucleotide is approx. 58 minutes, so long that the linear oligonucleotide is of little use for displacing one strand of the duplex target. However, the half-life of a double-stranded target in the presence of circular oligonucleotides of the invention is only 30 seconds.

Therefore, the circular oligonucleotides of the invention can be used not only to bind single-stranded targets but also to double-stranded targets. Accordingly, since both single-stranded and double-stranded nucleic acids can serve as targets for circular oligonucleotides, these circular oligonucleotides are more useful than linear oligonucleotides. For example, circular oligonucleotides of the invention are better regulators of biological processes in vivo and better in vitro diagnostic assays than corresponding linear oligonucleotides.

If the nucleic acid template extends beyond the central three-stranded target: a circle complex, a P or AP domain can be duplexed to either side of the standard triple complex. Thus, one target: a circular oligonucleotide complex can be partially double stranded and partly triple stranded, wherein the double stranded portions can be P: target duplexes without bound AP nucleotides or AP: target duplexes without bound P nucleotides. This layout is a tiered layout.

Each of the P domains, the AP domain and the target may independently have from 2 to 200 nucleotides, preferably from 4 to 100 nucleotides. The most preferred length is from 6 to 36 nucleotides.

The P and AP domains are separated by loop domains, which can independently have approx. 2 to 2000 nucleotides. The preferred loop length is 3-8 nucleotides, the most preferred loop being 5 nucleotides long.

According to the invention, loop domains do not have to be nucleotide bases. Non-nucleotide loops can make the production of circular oligonucleotides cheaper. More importantly, circular oligonucleotides having non-nucleotide loops are much more resistant to nucleases and therefore have a longer biological half-life than linear oligonucleotides. Further, uncharged loops or positively charged loops may promote binding by eliminating the negatively charged repulsion between the loop and the target. Further, uncharged or hydrophobic non-nucleotide circular oligonucleotides pass more easily through cellular membranes than circular oligonucleotides having a nucleotide loop.

As illustrated herein, non-nucleotide loop domains may consist of alkyl chains of polyethylene glycol or oligoethylene glycol chains or other chains, providing the required spherical or flexibility properties compatible with oligonucleotide synthesis. These chains have the same length of approx. They are 2-2000 nucleotides in length, preferably 3-8 nucleotides in length. The most preferred chain length for these chains is 5 nucleotides.

Preferred chains of non-nucleotide loop domains are polyethylene glycol or oligoethylene glycol chains. Preferably

27 oligoethylene glycol chains consist of chains similar to 5 nucleotides in length, e.g. preferably a pentaethylene glycol, a hexaethylene glycol or a heptaethylene glycol chain.

Circular oligonucleotides are single-stranded DNA or RNA, with nucleotides in guanine (G), adenine (A), thymine (T), cytosine (C) or uracil (U) bases, or any nucleotide analogue capable of forming hydrogen bonds in parallel cut in antiparallel fashion. Nucleotide analogs include: pseudocytidine, isopseudocytidine, 3-aminophenylimidazole, 2'-O-methyladenosine, 7-deazadenosine, 7-deazaguanosine, 4-acetylcitidine,

5- (Carboxy-hydroxylmethyl) -uridine, 2'-O-methylcytidine, 5-carboxymethylaminomethyl-2-thioridine, 5-carboxymethylaminomethyluridine, dihydrouridine, 2'-O-methyluridine, 2'-O-methyl-pseudouridine, beta , D-galactosylqueosine, 2'-O-methylguanosine, inosine, N6-isopentenyladenosine, 1-methyladenosine, 1-methylpseudouridine, 1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, 3- methylcitidine, 5-methyluridine, N6-methyladenosine,

-methylguanosine, 5-methylaminomethyluridine,

5-methoxyaminomethyl-2-thiouridine, beta-D-mannosilqueosin, 5-methoxycarbonylmethyluridine, 5-methoxyuridine, 2-methylthio-N6-isopentenyladenosine, N- (9-beta-D-ribofuranosyl-2-methylthiopurine-6-yl) ) carbamoyl) threone, N- (9-beta-D-ribofuranosylpurin-6-yl) methylcarbamoyl) threone. Where possible, either ribose or deoxyribose may be used with these analogs. Nucleotide bases in alpha-anomeric conformation may also be used in the circular oligonucleotides of the invention.

Preferred nucleotide analogs are unmodified

G, A, T, C, and U; pyrimidine analogs with lower alkyl, lower alkoxy, lower alkylamine, phenyl, or lower alkyl substituted phenyl at position 5 of the base, and purine analogs with similar groups at position 7 or 8 of the base. Particularly preferred nucleotide analogs are nucleotides having 2'-O-methylribose units instead of 5-methylcytosine, 5-methyluracil, diaminopurine and ribose or deoxyribose. As used herein, lower alkyl, lower alkoxy and lower alkylamine may have from 1 to 6 carbon atoms and may be straight or branched. These groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, amyl, hexyl and the like. The preferred alkyl group is methyl.

Circular oligonucleotides can first be generated as linear oligonucleotides and then converted to circular. Linear oligonucleotides can be prepared by any of several methods known in the art for preparing DNA or RNA oligonucleotides. For example, such methods include enzymatic synthesis and chemical synthesis.

Enzymatic methods for DNA oligonucleotide synthesis often utilize Klenow's, T7's, T4's Tag, or E. coli DNA polymerases according to Sambrook et al. . Enzymatic methods for RNA oligonucleotide synthesis frequently utilize SP6, T3 or T7 RNA polymerase as described in Sambrook et al.

. Reverse transcriptase can also be used for DNA synthesis from RNA (Sambrook et al.). The enzymatic production of the oligonucleotide requires a template nucleic acid which can be either chemically synthesized or obtained as mRNA as genomic DNA, cloned genomic DNA, cloned cDNA or other recombinant DNA. Some of the enzymatic methods for DNA oligonucleotide synthesis may require additional primary oligonucleotides that can be chemically synthesized. Finally, linear oligonucleotides can also be produced by PCR techniques as described e.g. Saiki et al. (1988, Science 239: 487).

The chemical synthesis of linear oligonucleotides is well known in the art and can be accomplished by solution or solid phase techniques. Furthermore, linear oligonucleotides having a defined sequence are commercially available or can be prepared by any of a variety of methods, including phosphoramidite, phosphite triester, H-phosphonate and phosphotriester, which are automated synthesis methods. The synthesis method chosen may depend on the length of the desired oligonucleotide and such choices are within the capabilities of one of ordinary skill in the art. For example, the phosphoramidite and phosphite triester method produces oligonucleotides containing 175 or more nucleotides, while the H-phosphonate method works well for oligonucleotides of less than 100 nucleotides. If modified bases occur in the oligonucleotide and preference is given to

Also, when modified phosphodiester bonds are used, the synthetic procedures may be modified according to known procedures. In this regard, Uhlmann et al.

(1990 Chemical Reviews 9-0: 543-584) provides information and outlines methods for preparing oligonucleotides with modified bases and modified phosphodiester bonds.

Synthetic linear oligonucleotides can be purified by polyacrylamide gel electrophoresis or by any of a number of chromatographic methods, including gel chromatography and HPLC. To verify a nucleotide sequence, the oligonucleotides may be subjected to DNA sequencing by any of the known methods, including the Maxam and Gilbert sequencing method, the Sanger sequencing method, the capillary electrophoretic sequencing method, the migratory point sequencing oligonucleotide sequencing method, by chemical degradation. The sequences of the short oligonucleotides can be analyzed by plasma desorption mass spectroscopy or by fast atom bombardment (McNeal, et al., 1982, J.Am.Chem.Soc. 104: 976; Viari et al., 1987, Biomed. Environ. Mass Spectrom., 14). : 83; Grotjahn et al., 1982, Nuc. Acid Res., 10: 4671). Sequencing methods are also available for RNA oligonucleotides.

The present invention provides a number of methods for producing circular oligonucleotides from linear precursors (e.g., loops), including the method of synthesizing a precursor and attaching it to an end-binding oligonucleotide and attaching the two ends of a lifetime. Any method for joining the two ends of an oligonucleotide is contemplated in the present invention, including chemical methods, e.g. known coupling agents such as BrCN, N-cyanoimidazole ZnC12, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and other carboimides and carbonyl diimidazoles are used. Further, the ends of a lifetime can be coupled by condensing a 5'phosphate and a 3'hydroxy or a 5'hydroxy and a 3'phosphate.

In accordance with the invention, there is also provided a simple one-step chemical method for constructing the circular oligonucleotides or circles of interest from the foreground. An oligonucleotide having the same sequence as the target nucleic acid was constructed; this is the end-linked oligonucleotide. A DNA or an RNA linear precursor is synthesized on a chemical or enzymatic residue and phosphorylated at its 5 'or 3' end, again on either a chemical or enzymatic residue. The precursor and the end-linked oligonucleotide are mixed and double-stranded to form a complex in which the 5 'and 3' ends of the lifespan are adjacent, as shown in Figure 2. It is preferred that the ends of the lifetime are located within a binding domain and not within the loop domain, preferably within the anti-parallel binding domain rather than within the parallel domain. Furthermore, it is preferable that the age of the patient has a 3'-phosphate instead of 5'-phosphate. After complex formation, the ends are one

undergo a condensation reaction in a buffered aqueous solution containing divalent metal ions and BrCN at about ph = 7.0. In a preferred embodiment, the buffer with imidazole-Cl 7.0 at pH 7.0 with divalent metal ions such as Ni, Zn or Co. Nickel is the most preferred divalent metal. Condensation occurs after about 6-48 hours after incubation at 4-37 ° C. Other divalent metals such as Cu, Pb, Ca and Mg may also be used.

By other methods of RNA circularization, the corresponding nucleotide sequences in the loop domain are preferably inserted into an RNA oligonucleotide to facilitate self-assembly, since the circular product is generated under appropriate conditions (Sugimoto et al., 1988, Biochemistry 27: 6384-6392). ).

Enzymatic encapsulation can also be performed using DNA ligase or RNA ligase under conditions appropriate to the enzyme.

Circular oligonucleotides may be separated from their templates by denaturing gel electrophoresis or melting followed by gel electrophoresis or other suitable chromatography or electrophoresis. The recovered circular oligonucleotide can then be further purified by standard techniques as required for use in the methods of the invention.

The invention illustrates the derivation or chemical modification of the oligonucleotide of interest by chemical moieties that promote cellular uptake. For example, covalent binding of a cholesterol moiety to an oligonucleotide can increase cellular uptake by 5 to 10-fold, which in turn increases DNA binding by 10-fold (Boutorin et al., 1989 FEBS Letters 254: 129-132). Other ligands may be useful as cellular receptors to enhance cellular uptake, e.g. insulin, transferrin and others. Similarly, derivatization of oligonucleotides with poly-L-lysine can facilitate cellular oligonucleotide uptake (Schell, 1974, Biochem. Biophys. Acta 340: and Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 648). Certain protein carriers may also promote cellular uptake, e.g. serum albumin, nuclear proteins that carry signals for transport to the nucleus, and viral or bacterial proteins that can penetrate the cell membrane. Therefore, protein carriers are useful when linked to or bound to the circular oligonucleotides of the invention. Accordingly, the invention illustrates the derivation of the circular oligonucleotides of interest with groups that promote cellular uptake; such groups include hydrocarbons and non-polar groups, cholesterol, poly-L-lysine and proteins, as well as other aryl or steroid groups and polycations which have analogous beneficial effects, e.g. phenyl or naphthyl groups, quinoline, anthracene or phenanthracene groups, fatty acids, fatty alcohols and sesquiterpenes, diterpenes and steroids.

The invention further illustrates the derivation of the oligonucleotides of interest by substances which are capable of cleaving or modifying the target nucleic acid or other nucleic acid strands that are related to or located near the target. For example, viral DNA or RNA can be targeted for destruction without damaging the cellular nucleic acid by adding a circular oligonucleotide complementary to the target nucleic acid that binds to a substance that can cleave or inactivate the viral DNA or RNA. -at. Nucleic acid damaging agents exemplified by the present invention for their cleavage or modifying activity include e.g. RNA and DNA nucleases, ribozymes that can cleave RNA, azidoproflavin, acridine, EDTA / Fe, chloroethylamine, azidofenacil and phenentroline / Cu. Uhlmann et al. (1990, Chemical Reviews 90:

543-584) provide further information on the use of such materials as well as oligonucleotide derivation methods that can be adapted to use the circular oligonucleotides of interest.

Derivation of the circular oligonucleotides of interest with moieties that promote cellular uptake or target binding, and nucleic acid derivatizing agents or drugs, can be accomplished by any of the methods known to those skilled in the art. Furthermore, the desired groups may be added to the nucleotides before synthesis of the oligonucleotides. For example, these groups can be linked to the T or C 5 positions and they are modified T and C. * ···.:. .. * ···

35 nucleotides may be used to synthesize circular oligonucleotides of the invention. In addition, derivation of the selected nucleotides allows insertion of the group into the circular oligonucleotide selected domain. For example, in some cases, it is preferable for the moieties to be inserted into a loop where the moiety does not act on the bond or the AP or P domain to facilitate cleavage or modification of the target nucleic acid.

In accordance with the invention, modification of the phosphodiester backbone of circular oligonucleotides has also been illustrated. Such modifications may promote oligonucleotide uptake by cells or extend the biological half-life of such nucleotides. For example, circular oligonucleotides can enter the cell much more quickly if the negative charge from the internucleotide phosphate is removed. This can be accomplished by replacing the negatively charged oxygen of the phosphate with a methyl group, an amine, or by replacing the phosphodiester bond with a phosphotriester bond by adding an alkyl group to the negatively charged phosphate oxygen. Alternatively, one or more of the phosphate atoms that are involved in the normal phosphodiester bond are exchanged. For example, NH-P, CH ₂ ~ P or SP bonds can be formed. Accordingly, the present invention relates to the use of methylphosphonate, phosphorothioate, phosphorodithioate, phosphotriester and phosphorus boron bonds (Sood et al., 1990, J. Am. Chem. Soc. 112: 9000). The phosphodiester group may be replaced by siloxane, carbonate, acetamidate or thioether groups. These changes may also enhance the issue in question

4 'oligonucleotide resistance to nucleases. Methods for the synthesis of modified phosphodiester-linked oligonucleotides are reviewed in Uhlmann et al.

Circular oligonucleotides having non-nucleotide loops may be prepared by any of the known methods. For example, Durand et al. (1990, Nucleic Acids Sl. 18: 6353-6359) provide synthetic methods for binding non-nucleotide chains to DNA. Such methods can generally be adapted to allow for the automated synthesis of a linear oligonucleotide precursor which can then be used to produce the circular oligonucleotide of the invention. In general, in standard DNA synthesis, groups reacting with nucleotides, e.g. phosphoramidite, H-phosphonate, dimethoxytrityl, monomethoxytrityl and the like can be placed at the ends of the non-nucleotide chains and the nucleotides corresponding to the ends of the P and AP domains can be attached.

Further, various nucleotide sugars can be incorporated into the oligonucleotides of the invention. For example, RNA oligonucleotides may be used because RNA: DNA hybrids are much more stable than DNA: DNA hybrids. Further binding stability can be achieved by using 2'-O-methyl ribose in the circular oligonucleotides of the invention. Phosphoramidite chemistry can be used to synthesize RNA oligonucleotides as described (Reese, CB In: Nucleic Acids & Molecular Biology; Springer-Verlag: Berlin, 1989; Vol. 3, p. 164; and Rao et al., 1987, Tetrahedron Lett. 28). : 4897).

Synthesis of RNA 2'-O-methyl oligoribonucleos and nucleotides oligo37 DNA is only slightly different. RNA 2'-O-methyloligonucleotides may be prepared by minor modifications of the amidite, H-phosphonate or phosphotriester methods (Shibahara et al., 1987, Nucleic Acids Slide 15: 4403; Shibahara et al. 1989, Nucleic Acids Slide 17: 239). , Anoue et al., 1987, Nucleic Acids Res. 15: 6131).

In another embodiment of the invention, circular oligonucleotides can accelerate dissociation of double-stranded nucleic acid targets. Therefore, double-stranded nucleic acid targets need not be subjected to denaturing conditions prior to binding to the circular oligonucleotides in question. Thus, circular oligonucleotides can bind to both single-stranded and double-stranded nucleic acid targets under a wider variety of conditions, preferably physiological conditions. The circular oligonucleotides of the invention are several orders of magnitude faster in accelerating the displacement of duplex nucleic acid strands than the corresponding linear oligonucleotides.

The present invention therefore provides a means for displacing one strand of a double-stranded nucleic acid target with one of the circular oligonucleotides of interest without prior denaturation of the double-stranded nucleic acid target. Thus, the invention provides a method for displacing a single strand of a double-stranded nucleic acid by contacting the target with one of the circular oligonucleotides of interest for a specified period of time and under conditions which permit the binding of the circular oligonucleotide to the target. The circular oligonucleotides of interest may be targets of a double-stranded nucleic acid, either RNA or DNA, which has not undergone denaturation e.g. heat treatment or exposure to alkaline pH.

According to the application, the nucleic acids for fiber motility may be present in an organism or in a sample containing a pure or non-pure nucleic acid composition, a piece of tissue, a prokaryote or a eukaryotic smear, a chromosomal mass, and the like. In addition, the nucleic acid targets used for strand motifs with the present circular oligonucleotides include viral, bacterial, fungal, or mammalian nucleic acids.

According to the invention, conditions effective for denaturing the target strand by displacement, which then allow binding, include the appropriate circular oligonucleotide to target nucleic acid ratio. In addition, for use in the present invention, the appropriate circular oligonucleotide: target ratio is about 1: 100, preferably 1:50.

Furthermore, according to the invention, the effective time for denaturation of the double-stranded nucleic acid achieved by oligonucleotide displacement by the invention is from about 1 minute to about 1 minute. 16 o'clock.

A circular oligonucleotide may first bind to the P domain for a duplex purpose. Binding to such a P domain puts the nucleotides of the AP domain into a juxta position to compete for Watson-Crick binding to the target nucleotide. This P domain pre-attachment, followed by competition for the Watson-Crick binding of the AP domain nucleotide, can provide the bases of acceleration observed in the strand displacement generated by circular oligonucleotides.

In summary, the circular oligonucleotides of the invention have three important properties that allow strand displacement. First, the circular oligonucleotide has the ability to pre-attach, resulting in high local concentrations. Second, the circular oligonucleotide contains a second (AP) binding domain that competes for binding to a complementary strand of a duplex. Finally, the circular oligonucleotide binds with greater affinity than the displaced strand of the duplex, thus completing the reaction.

The present invention addresses variations in the applicability of the circular oligonucleotides of interest which are made possible by their selective and stable binding properties to single-stranded and double-stranded targets. Uses include, but are not limited to: the use of solid sequence-linked circular oligonucleotides for affinity isolation of complementary nucleic acids; using the oligonucleotides of interest to provide sequence-specific stop signals for polymerase chain reactions (PCR); covalently linking drugs, drug analogs, or other therapeutic agents to circular oligonucleotides to allow cell type-specific drug delivery; labeling of circular oligonucleotides with a detectable reporter molecule for the purpose of lysing, quantifying, or identifying the complementary target nucleic acid locus; and the binding of circular oligonucleotides to a cellular or viral nucleic acid template and the regulation of biosynthesis directed by that template.

Circular oligonucleotides can be coupled to solid supports such as silica gel, cellulose, nylon, and other natural and synthetic materials that are used to make beads, filters, and column chromatography resins. Methods for attaching nucleic acids to such solid supports are well known; any known connection method is contemplated in the present invention. A circular oligonucleotide linked to a solid support can be used to isolate a complementary nucleic acid. Isolation of the complementary nucleic acid can be accomplished by inserting the oligonucleotide: solid support into a column for chromatography. Other methods of isolation may be performed without inserting the oligonucleotide: solid support into a column, e.g. using filtration methods. The circular oligonucleotide: solid support, for example, can be used to isolate poly (A) ⁺ mRNA from total cellular or viral RNA by generating a circular oligonucleotide having a poly (dT) or poly (U) domain in the P and AP domains. Circular oligonucleotides are ideally suited for this type of application as they are nuclease resistant and very highly bound to the target nucleic acids.

Other applications of the circular oligonucleotides in question are in the field of polymerase chain reaction (PCR) technology. PCR technology provides methods for synthesizing a double-stranded DNA fragment encoded between two known nucleic acid sequences used as the primary binding site. In some cases, it is desirable that the single-stranded DNA fragment be generated before or after the double-stranded fragment. This can be done e.g. by binding a circular oligonucleotide of the invention to one of the primary binding sites or to a site lying between the primary binding sites.

The present invention relates to the use of circular oligonucleotides for targeting drugs against specific target cells. Such control may allow selective disruption or amplification of certain cell types e.g. inhibition of tumor cell proliferation is achieved. Different cell types express different genes, so the concentration of a particular mRNA may be higher in one cell type than in another, such mRNA target mRNA being the cell type specific drug delivery by a circular oligonucleotide bound to drugs or drug analogs. Cells containing a high concentration of the target mRNA are made targets of drug delivery by adding to the cell a circular oligonucleotide covalently linked to a drug complementary to the target mRNA.

The present invention relates to the labeling of circular oligonucleotides of interest for use as a probe for the detection of a target nucleic acid. The labeled circular oligonucleotide

- 42 probes can be used for diagnostic and analytical hybridization procedures for the localization, quantification or detection of the target nucleic acid in tissue, chromosomes or nucleic acid mixtures. Circular oligonucleotide probes of the invention represent a fundamental improvement over linear nucleic acid probes, since circular oligonucleotides can displace one strand of the double-stranded nucleic acid, and because the oligonucleotides have two binding domains that not only provide increased binding stability, : oligonucleotide interaction.

Labeling of a circular oligonucleotide can be accomplished by inserting a nucleotide bound to a reporter molecule into the circular oligonucleotide of interest. The reporter molecule for use in the present invention is a molecule or atom which by its chemical nature provides an identifiable signal for detection of a circular oligonucleotide. This detection can be quantitative or qualitative. The invention relates to any commonly used reporter molecule, including radionuclides, enzymes, biotins, psoralens, fluorophores, chelated heavy metals, and luciferin. The most commonly used reporter molecules are either enzymes, fluorophores, or radionuclides that are linked to nucleotides involved in circular oligonucleotide synthesis. Commonly used enzymes include horseradish peroxidase, alkaline phosphatase, glucose

- 43 oxidases, and beta-galactosidase, among others. Substrates used in combination with specific enzymes are generally selected because a detectable color product is formed during the action of the enzyme acting on the substrate. For example, p-nitrophenyl phosphate can be suitably used with alkaline phosphatase conjugates, 1,2-phenylenediamine, 5-aminosalicylic acid or toulidine are generally used for horseradish peroxidase. The probes thus created can be used to detect a specific DNA or RNA target, e.g.

Southern analysis, Northern analysis, in situ hybridization to a piece of tissue or chromosome mass and other analytical or diagnostic procedures. Methods employing such hybridization probes are well known and are exemplified by Sambrook et al.

The circular oligonucleotides of the invention may be used in agreement with any known detection or diagnostic procedure based on the hybridization of a probe target to a nucleic acid. Further, the circular oligonucleotides of the invention can be used in any hybridization method that quantifies a target nucleic acid, e.g. by competitive hybridization between a target nucleic acid in a sample and a labeled target for one of the present oligonucleotides. Further, reagents for the preparation of a circular oligonucleotide probe and for use in such a probe hybridization process may be commercially available in a kit.

The kit may be divided into compartments for ease of use and may include at least one first compartment, one

reagents for producing a circular oligonucleotide precursor to life, at least a second compartment providing reagents for labeling the reporter molecule with a lifetime, at least a third compartment providing reagents for circulating age, and at least a fourth compartment for isolating labeled circular oligonucleotides.

The invention further provides a kit for isolating a template nucleic acid. Such a kit has at least one first compartment that provides a circular oligonucleotide that is complementary to the target within the template. For example, the template nucleic acid may be a cellular and / or viral poly (A) ⁺ mRNA and the target may be a poly (A) ⁺ tail. However, the circular oligonucleotides of the invention - useful in isolating poly (A) ⁺ mRNA - have the sequences of the poly (dT) or poly (U) P and AP domains.

In addition, the invention provides kits useful when the diagnosis of a disease depends on the detection of a specific known target nucleic acid. Such nucleic acid targets may be e.g. a viral nucleic acid, an extra or a missing chromosome or gene, a mutant cellular gene or chromosome, an aberrated RNA, or others. The kit may be divided into compartments to include at least a first compartment providing a circular oligonucleotide bound to a reporter molecule and at least a second compartment providing reagents for detecting the reporter molecule.

In one aspect of the invention, a DNA provides a method of regulating RNA or protein biosynthesis by contacting at least one of the circular oligonucleotides of interest with a template of said DNA, this RNA, or a nucleic acid of said protein in an amount and under conditions satisfactory to the oligonucleotide. leotide (s) for binding to the target sequence (s) in the template. Binding between the oligonucleotide (s) and the target inhibits access to the template and thereby regulates the biosynthesis of the nucleic acid or protein. Blocking access to the template protects proteins and nucleic acids involved in the biosynthetic pathway from binding to the template, moving on the template, or recognizing the signals encoded in the template. Alternatively, if the template is an RNA, regulation may involve allowing selective degradation of the template. For example, the RNA templates bound by the circular oligonucleotides in question are sensitive to degradation by RNase H, and degradation of the selected RNA template by RNase H may thus regulate the use of the template in biosynthetic processes.

For the purposes of the present invention, the biosynthesis of a nucleic acid or a protein includes cellular and viral processes such as cellular and viral processes. DNA replication, DNA reverse transcription, RNA transcription, RNA assembly, RNA polyadenylation, RNA translocation and protein translation and those that lead to the production of DNA, RNA and protein and involve a biosynthetic process in some phases

- 46 nucleic acid templates included.

In the use of the present invention, control of biosynthesis includes inhibiting, stopping, enhancing, accelerating or delaying biosynthesis. The regulation can be direct or indirect, e.g. the biosynthesis of a DNA, RNA or protein can be directly controlled by binding a circular oligonucleotide to a template of that DNA, RNA or protein; or alternatively, biosynthesis can be indirectly controlled by binding the oligonucleotide to a second template encoding a protein involved in the biosynthesis of the first DNA, RNA or protein.

Nucleic acid therapies may be RNA or DNA and may be single-stranded or double-stranded. While the circular oligonucleotides of the invention bind to only one strand of the target present in the template, the double-stranded templates are open during the biosynthetic pathway and will therefore be available for binding. In addition, the P domain of the circular oligonucleotides of the invention may bind to a double-stranded target and place the AP domain in a position to compete for Watson-Crick binding to the target nucleotide.

DNA replication from a DNA template is mediated by proteins that bind to an origin of replication, where they open DNA and initiate DNA synthesis along the DNA template. To inhibit DNA replication in accordance with the invention, circular oligonucleotides that bind to one or more targets in a replication origin are selected. The

- 47 · Such binding prevents template proteins that are involved in DNA replication from being accessible to the template. Therefore, initiation and continuation of DNA replication is inhibited. Another method of inhibiting DNA replication is by inhibiting the expression of proteins involved in mediating DNA replication, e.g. at the transcriptional or translational level.

DNA replication from the RNA template is mediated by a reverse transcriptase bound to an RNA region that binds to a nucleic acid printer itself. To inhibit DNA replication from the RNA template, the binding of the reverse transcriptase or the primer can be inhibited by binding a circular oligonucleotide to the primary binding site, thereby inhibiting access to the site. In addition, inhibition of DNA replication may occur by binding a circular oligonucleotide to a site in an RNA template, since such a binding can inhibit this site and downstream sites, e.g. accessibility to sites on the 3 'side of the target or binding site.

To initiate RNA transcription, RNA polymerase recognizes and binds to specific start sequences or promoters on the DNA template. Binding of RNA polymerase opens the DNA template. There are additional transcriptional regulatory elements that play a role in transcription and are located on the DNA template. These transcription elements include enhancer sequences, upstream activation sequences, repressor binding sites, and others. All of these promoters and transcriptional controls are alone

In 48 combinations, the invention is directed to circular oligonucleotides. Oligonucleotides that bind to these sites can block access to the template for RNA polymerase and transcription factors, and thereby regulate, i.e., increase or decrease RNA production, particularly mRNA and tRNA. Furthermore, the oligonucleotides of the invention may target the template coding region or the 3'-untranslated region of DNA, thereby causing premature termination of transcription. One skilled in the art will readily design oligonucleotides for the above target sequences from known sequences of these regulatory elements, coding region sequences, and consensus sequences.

RNA transcription can be enhanced, e.g. by binding a circular oligonucleotide to a negative transcriptional regulatory element, or by inhibiting the biosynthesis of a protein that may repress transcription. Negative transcriptional regulatory elements include repressor sites or operator sites in which a repressor protein binds and blocks transcription. Oligonucleotides that bind to repressor or operator sites can block the access of repressor proteins to their binding sites and thereby increase transcription.

Primary RNA or pre-mRNA produced in eukaryotic cells undergoes several maturation processes before being translocated in the cytoplasm for translation of the protein. Introns are removed from the premRNA in the nucleus during assembly reactions.

···· • · ·

The 5 'end of the mRNA is modified to take up the 5' cap structure, thereby stabilizing the mRNA. Many bases are also changing. Polyadenylation at the 3 'end of the mRNA is believed to involve delivery from the nucleus. The circular oligonucleotides of the invention may block any of these processes.

The pre-mRNA template is assembled in the nucleus by ribonucleoproteins that bind to assembly points and in the pre-mRNA by intron splice point sequences. The consensus sequences of the 5 'and 3' assembly points and the intron branching points are known. For example, inhibition of binding of ribonucleoproteins to assembly points or inhibition of covalent binding of the 5 'end of the intron to the intron junction point may block assembly. Therefore, maturation of the pre-mRNA template can be blocked by preventing access to these sites, i.e., the circular oligonucleotides of the invention can be linked to a 5 'binding site, an intron junction site, or a 3' binding site. Assembly of a specific pre-mRNA template can be inhibited by the use of circular oligonucleotides with sequences complementary to the specific pre-mRNA assembly site (s) or the intron junction. In a further embodiment, assembly of the linked, pre-mRNA assembled templates can be inhibited by a mixture of circular oligonucleotides having sequence variants that, when used together, are complementary to the desired set of assembly sites and intron branching sequences. · · ·

Λ ·· «4 ··» hrs.

Polyadenylation involves the recognition of a pre-mRNA and cleavage of a specific RNA by endonuclease at specific polyadenylation sites, followed by the addition of a poly (A) tail to the 3 'end of the pre-mRNA. However, any of these steps can be inhibited by binding the circular oligonucleotides of interest to appropriate sites.

A poly (A) tail appears to be required for translocation of RNA from the nucleus to the cytoplasm in eukaryotic cells. Thus, in accordance with the invention, a circular oligonucleotide was designed which binds to the poly (A) tail and thereby inhibits access to the poly (A) tail and inhibits RNA translocation. For such an oligonucleotide, both the P and AP domains may contain about 10-50 thiamine residues, preferably about 10 to about thiamine residues. 20 leftovers. Particularly preferred lengths of the P and AP domains of the oligonucleotide represent about 6-12 thiamine residues.

Protein biosynthesis begins by binding the ribosomes to an RNA template, followed by the initiation and extension of the amino acid chain by translational reading of the mRNA. Thus, protein biosynthesis or translation can be inhibited by inhibiting template access by using the circular oligonucleotide of interest to bind to targets in the template mRNA. Such targets contemplated by the present invention include ribosome binding sites (Shine-Delgarno sequence), a 5'mRNA cap site, an initiation codon, and protein coding sequence sites. There is a class of proteins which divide into the homologous domains of a nucleotide sequence. Thus, inhibition of the biosynthesis of proteins of this class can be achieved by targeting homologous protein domains (via the coding sequence) to the circular oligonucleotides of interest.

Controlling biosynthesis by any of the aforementioned methods has many applications. For example, genetic disorders can be corrected by inhibiting the production of mutant or overproduced proteins, or by increasing the production of under-expressed proteins, genes encoding factors that regulate cell proliferation, such as the spread of cancer and the inhibition of virally encoded functions. for the purpose.

Some types of genetic disorders that can be treated with the circular oligonucleotides of the invention include Alzheimer's disease, some types of arthritis, sickle cell anemia, and others. Numerous viral infections can be treated using the circular oligonucleotides of the invention, including influenza, rhinovirus, HIV, herpes simplex, papilloma virus, cytomegalovirus, Epstein-Barr virus, adenovirus, vesticular stomatitis virus, rotavirus, and others. infections. Animal and plant viral infections of the present invention may also be treated using the oligonucleotides of interest.

The c-myc gene is an example of a gene that plays a role in cell proliferation. Inhibition of c-myc expression has been demonstrated in.

·· ····

52 in vitro using a linear oligonucleotide complementary to a 115 bp upstream c-myc transcription start site (Cooney et al., 1988, Science, 241: 456-459). The circular oligonucleotides of SEQ ID NO: 0.1 and SEQ ID NO: 2, as described below, are complementary to the c-myc promoter at nucleotides -131-120 and -75-62, respectively, and in accordance with the invention. c-mvc expression. As used in the descriptions of SEQ ID NO: 1 and SEQ ID NO: 2, N can be any nucleotide or nucleotide analog.

SEQ ID NO: 1

N C T C C C C G C C C T C N N N N N N N N C T C C C C THE C C C T C N

SEQ ID NO: 2

N T C T T T T T T c T T T T C N N N N N N N N T C T T T T T T c T T T T C N

Human Immunodeficiency Virus (HIV) is a retrovirus that causes acquired immune deficiency syndrome (AIDS). The circular oligonucleotides of the present invention provide a means of blocking the replication of this virus without adversely affecting normal cellular replication in humans infected with HIV. The retroviral genome is transcribed as a single, long transcript, some of which is assembled to generate RNA encoding viral coat proteins. Inhibition of HIV infection can be achieved by designing oligonucleotides related to multiple regions within the HVV genome. These regions include regions encoding functions for genomic replication (e.g., pol or reverse transcriptase function) or functions that regulate gene expression (e.g., tat, reverse or other functions). However, previous work with linear oligonucleotides has suggested that assembly sites, poly (A) addition signals, cap or start codon sites, and sites involved in ribosome assembly may be particularly effective in inhibiting eukaryotic protein expression. Furthermore, the retroviral genomic terminal structure is also an excellent target for inhibition of retroviral production, not only because these constructs encode regulatory regions that influence the rate of transcription and replication, but also because these constructs allow repetition and oligonucleotide binds and blocks access to each repeat region.

Accordingly, the invention provides two circular oligonucleotides disclosed in SEQ ID NO: 3 and SEQ ID NO: 4, wherein N may be any nucleotide or nucleotide analog and Y may be a pyrimidine or a pyrimidine analog. SEQ ID NO: 3 is complementary to an HIV-1 binding site (6039-52 nucleotides), and SEQ ID NO: 4 is complementary to a portion of the tat gene (5974-88 nucleotides). The circular form of SEQ ID NO: 3 is shown below, wherein nucleotide 1 is the first nucleotide in the P domain, e.g. the first T in the first row is »

The circular form of SEQ ID NO: 4 is shown below, wherein nucleotide number 1 represents the first nucleotide of the P domain.

SEQ ID NO: 3 and

SEQ

Circular oligonucleotides ID NO: 4 can inhibit Hiv infection both in vitro and in vivo. In screening the efficacy of circular oligonucleotide against HIV infection, one of ordinary skill in the art will be able to demonstrate in vitro stability of the oligonucleotide: target binding and to evaluate in vivo efficacy and binding stability. To monitor in vitro inhibition, circular oligonucleotides may be added to the growth medium of an appropriate HIV-infected cell line. Cells may be pretreated with circular oligonucleotides, or circular oligonucleotides may be added to the cells at the time of infection or after HIV infection. Addition before or after infection allows to determine whether the oligonucleotide in question can prevent or merely prevent Hiv infection.

The degree of inhibition of HIV infection or inhibition of replication can be estimated by a variety of assay methods, including: estimating the survival rate of oligopnucleotide-treated cells relative to untreated cells; quantification of viral antigen produced in treated and untreated cells.

In vivo testing of the efficacy of circular oligonucleotides may be performed in a suitable animal host, e.g. transgenic mice or chimpanzees. Levels of HIV antigens can be monitored to estimate the effect of circular oligonucleotides on HIV replication, thus providing an opportunity to follow the disease process. Alternatively, human volunteers with AIDS or ARC may be treated with the circular oligonucleotides of interest since the oligonucleotides do not appear to be cytotoxic. The disease status of these volunteers can be assessed to determine how effective the oligonucleotides in question are in treating and preventing the AIDS infection.

In another embodiment, the invention provides pharmaceutical compositions comprising the circular oligonucleotides of interest in association with a pharmaceutically acceptable carrier. Preferably, the oligonucleotides of interest are present in about one to three minutes. 0.1 µg to 100 mg / kg body weight per day in a therapeutically effective amount, more preferably about 10 µg / kg body weight / day. 0.1 µg to about 10 mg / kg body weight / day for binding to its nucleic acid in accordance with the methods provided by the invention. Dosages can be readily determined by one of ordinary skill in the art and formulated in the pharmaceutical composition in question.

For the purposes of the present invention, a pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such materials and media as pharmaceutically active substances is well known in the art. Any conventional medium or substance compatible with its active ingredients is contemplated for use in a therapeutic composition. Additional active ingredients may also be incorporated into the composition.

The oligonucleotides of interest are administered topically or parenterally, e.g. intravenously, intramuscularly, intraperitoneally, subcutaneously, or intradermally, or when properly protected, the oligonucleotides of interest may be administered orally. The oligonucleotides in question may be in a solution or suspension in a cream in the form of a solution or a suspension.

- 58 for local applications. For oral administration, the oligonucleotides can be protected by encapsulation in a gelatin capsule. The oligonucleotides may be embedded in liposomes or polyethylene glycol-modified liposomes for parenteral administration. Incorporation of additional substances into liposomes e.g. Antibodies reactive against membrane proteins found on specific target cells can help to target oligonucleotides to specific cell types.

Topical and parenteral administration in a liposome carrier is the preferred application.

The following examples further illustrate the invention.

Example 1

Circularization of oligonucleotides using an end-linked oligonucleotide

In accordance with the present invention, a simple one-step chemical method has been developed for constructing circles from linear precursors. A DNA oligonucleotide having the same sequence as the final target was constructed, the end-linked oligonucleotide. A lifetime oligonucleotide was then generated and chemically phosphorylated at the 5 'or 3' ends. As shown in Fig. 2, the precursor and the end-linked oligonucleotide were mixed and allowed to form a complex in which the ends are adjacent. Cyanogen bromide, imidazole buffer and a divalent metal were added. After incubation for 6-48 hours, the mixture was dialyzed, lyophilized, and the products were separated by 20% polyacrylamide gel electrophoresis denaturation. UV illumination covered the major bands that migrated together with the forebrain and end-linked oligonucleotide, and with a new product that migrated a little slower than the living age. No product was observed if the final oligonucleotide was not added to the mixture or if the 5'- or 3'-phosphate group was missing from the foreground. The major bands were excised and eluted from the gel, dialyzed to remove salts, and quantitated by measuring absorbance at 260 nm. For reactions with Primer 1 and 2 (SEQ ID NO: 5 and SEQ ID NO: 6, respectively) using end-linked oligonucleotides 4 and 5 (SEQ ID NO: 8 and SEQ ID NO: : 9, respectively), we obtained Circles 6 and 7 with 40% and 58% yields, respectively.

The sequences of these molecules and other oligonucleotides are shown in Figure 3.

The circular structure of products 6 and 7 was evidenced by its resistance to 3 'exonuclease digestion and 5' dephosphorylation under conditions in which the linear lifetime was completely destroyed or dephosphorylated. Accordingly, the 3 'exonuclease activity of the T4 DNA polymerase cleaved linear precursors 1 and 2, but not circles 6 and 7. The linear curves were labeled at their 5 'end with ³² P and circularized. After reaction, the circular products were inert to calf alkaline fősz60 ligation reaction, while the areas produced completely liberated the ²² P labeling. The slightly slower gel mobility of the circles compared to the circles was similar to that of circularization. The optimum conditions for circularization are given below.

A number of parameters have been optimized to increase the yield of the circular product, including oligonucleotide and age concentration, temperature, reaction time, metal, metal concentration, BrCN concentration and pH. Providing the circularization conditions developed resulted in the formation of at least twice the circular product compared to prior art conditions in which two single-stranded oligonucleotides were linked (Luebke et al., 1989, J. Am. Chem. Soc. 111: 8733 and Kanaya et al., 1986, Biochemistry 25: 7423).

These developed circumstances are:

uM age-specific uM terminal oligonucleotide

100 mM NiCl2

200 mM imidazole-HCl (pH 7.0)

125 mM BrCN

C, 36 hours

However, closing the circle was also effective in the following circumstances:

- 61 Other metals

3-200 M élőkor 3-300 M end brace solos oligonucleotide 10-500 mM NiC12 50-500 mM imidazole-HCl 20-200 mM BrCN (Zn ^{2 +} ; Mn ²⁺ ; Co ²⁺ ; Cu ²⁺ ; Pb ²⁺ ; Ca ²⁺ ; mg

²⁺ ) also worked instead of Ni ²⁺ . Furthermore, the reaction is pH sensitive.

The closure of the AP and P domains is described below.

Closure of a circle in the AP domain was superior to closure in the P domain. Comparison of the circularization of precursors 2 and 3 (SEQ ID NO: 6 and SEQ ID NO: 7, respectively) around the same end-linked oligonucleotide (e.g., 5, SEQ ID NO: 9) showed that Circle 7 (having SEQ ID N0: 6) was created in 58% when closed in AP domain (e.g. using precursor 2) and only in 35% when closed in domain P (for example, using precursor 3).

The condensing reagents are described below

Two commonly used reagents for chemical ligation of DNA and RNA, BrCN / imidazole / NiCl2 and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) (Kanaya et al., 1986, Biochemistry. 25: 7423 and Ashley et al., 1991, Biochemistry 30: 2927). Therefore, these reagents were directly compared for the efficacy of ligation to a lifetime circular oligonucleotide 6 (Figure 3 and SEQ ID NO: 5) with a dA (SEQ ID NO: 5). ID NO: 8) using an end-linked oligonucleotide.

The BrCN / imidazole / NiC12 ^- was used in the indicated optimal conditions except that ligation efficiency was observed at 4 ° C and the 25th EDC was used at 200 mM concentration with 20 mM MgCl ₂ , 50 mM MES (pH 6.0) at 4 and 25 ° C for 4 days.

BrCN was much more efficient at 4 ° C, yielding 95% circular product, while EDC only 55%. However, at 25 ° C, both EDC and BrCN gave 95% product. So BrCN is much more efficient at lower temperatures, but EDC and BrCN can be used with equal chances at 25C. However, BrCn has an additional advantage over EDC, as it requires 24 hours or less to ligate with BrCN, and about 4 days to ligate with EDC.

The use of 5 'or 3' phosphate is described below

Under different ligation conditions, the coupling of a 3'-phosphate with 5'-0H resulted in more ligated products than the coupling of a 5'-phosphate with 3'-0H (Ashley et al.,)

Therefore, 5'-phosphate or 3'-phosphate are provided with precursors

The percent conversion to circular oligonucleotide 6 (Figure 3) was compared:

• · ·· • · ♦ ·· ··· • «· · · ♦ · · · ·

Γ £ ΐ 'ΤΊ'Ί'Ί * TT? T ΤΤ

C

Α

C

Α

C

C

Α

C

Α

C ττττττ ττττττ

ΗΟ ΟΡΟ3 = ττττττττττττ

C

Α

C

Α

C

C

Α

C

Α

C ττττττ ττττττ = Ο3ΡΟ ΟΗ

The circularization reaction was performed using dA ₁₂ end-linked oligonucleotide under the given optimum conditions, except that 5 nmol precursor and end-linked oligonucleotide were used. The product was visualized under UV light after separation by <RTI ID = 0.0> denaturing </RTI> by gel electrophoresis.

Conversion to circular product was 60% (+, - 5%) when 5'-phosphate was present and 95% if 3'-phosphate was present. No increase in yield was observed when reaction time was increased or reagent concentrations were increased.

Accordingly, the use of 3'-phosphate increases circularization rather than the application of 5'-phosphate.

Example 2

Circular oligonucleotides bind to the target nucleic acids with greater affinity than linear oligonucleotides

The binding affinity of circles 6 and 7 (SEQ ID NO: 5 and SEQ ID NO: 6, respectively) for their targets was determined by comparing the melting temperatures of the circular and linear complexes. The solutions of the oligonucleotides and the target (3 uM each) in 1: 1 ratio contained 100 mM NaC12 ^_ in 10 mM Tris-HCl (pH 7.0). Mixing curves (measured at 260 nm) confirmed the formation of complexes at a 1: 1 ratio. The free energy (- delta G37) of the complexes was obtained from the melting data using a two-step curve fitting method (Petersheim et al., 1983, Biochemistry 22: 256).

The results showed that circular oligonucleotides bind to their targets much more strongly than linear primers or Watson-Crick complementary target sized oligonucleotides (♦ · * · ( Table 2). For example, with Watson-Crick complement of target size 4 (SEQ ID NO: 8), duplexes were created with a T _m of 37.1 C, while at age 1: target complex 4 (e.g., SEQ ID NO: 5, by SEQ ID NO: 8 binds) had 44.7 CT value _m. By way of comparison, Round 6, having the same sequence as Lifetime 1, bound to Target 4 with a value of 57.5 CT _m and the free energy of binding was 8.6 kcal / mol, much more advantageous than the corresponding Watson-Crick duplex. The corresponding association constant at 37 ° C is 6 x 10 ¹¹ M ^-1 , which is 6 orders of magnitude higher than that of the Watson-Crick duplexes. A similar effect was observed upon binding of circle 7 (SEQ ID NO: 6) to target 5 (SEQ ID NO: 9); this complex has a T _m of 62.3 ° C, while the corresponding Watson-Crick duplex melts at 43.8 ° C. These data indicate that circular oligonucleotides bind more strongly to a target than linear oligonucleotides.

To determine binding properties when the target sequence is embedded in a longer sequence, an oligonucleotide of 36 nucleotides in length is synthesized with a 12 base target sequence (corresponding to target 4). Melting studies revealed that ring 6 was much more strongly bound to this longer oligonucleotide than the target of the same size as the ring binding domain: ring 6 at target 4 had a T _m of 59.4 C, while embedded target comprising 36 base oligonucleotide _Tm

63.4 ° C. Therefore, the binding strength of circles with embedded targets was greater than those with embedded domain-sized targets.

To test the binding binding affinity of round 6 RNA, the oligoribonucleotide rA _{12 was} synthesized and the T _m value of round 6 rA _{12 was} determined. Circuit 6 had a T _m of rA ₁ ^_ 2 V of 58.3 ° C relative to dA ₁ 2 ^_ of 57.8 CT _m . These data show that circles are bound to RNA targets just as strongly, if not more strongly, than to DNA targets.

- 67 «« ·

TABLE II Oligonucleotide:

target complex ^T m ' ^c -G37 (kcal / mol) ~ ^j TTTTTTTTTTTT

37.1

8.1

5'-AAAAAAAAAAAA

3'-TTCTTTTCTTTC

43.8

10.3

5'-AAGAAAAGAAAG

T 'TTTTTT T ΤΊ * Ί' Γ

1: 4

C AAAAAAAAAAAA

44.7

10.5

T'T'TT'TT * TTTTTT

OPO3 = • · «« ·· ·· ♦ • · ···· · ·· *

• * ·

3: 5

TTCTTTTCTTTC

C AAGAAAAGAAAG C

TTCTTT TCTTTC

OPO3 =

II. Resume table

TTTTTTTTTTTT

6: 4

C AAAAAAAAAAAA

57.4

16.7 ^ί ΓΤ ^, Γ ^Γ ΓΤ? Ί ^1, Ι ^, ΤΤΤΤΤ

TTCTTTTCTTTC

C AAGAAAAGAAAG

62.3

16.4

TTCTTTTCTTTC

Example 3

Circular oligonucleotides bind to the target much more selectively than linear oligonucleotides

To determine the sequence selectivity of circular oligonucleotides, a series of target oligonucleotides was generated with a variable base. The binding energy of the complex formation of a circle for these purposes was measured and the selectivity was determined from the free energy differences between the correct sequence and the mismatched sequence. The selectivity obtained with the circular structure was directly compared with the selectivity of an analog linear oligonucleotide.

The DNA oligonucleotides were machine-synthesized using the beta-cyanoethyl phosphoramidite method. Circular oligonucleotide 8 with SEQ ID NO: 7

5'-pTCTTTCCACACCTTTCTTTTCTTCACACTTCTTT was prepared from a linear precursor having an end-linking oligonucleotide having the sequence: 5'AAGAAAAGAAAG (SEQ ID NO: 9) using BrCN / Imidazole as described in Example 1 for closing the final bond. The circular structure was confirmed to be resistant to 3'exonuclease and 5'-phosphatase.

The selectivity of the 8-round sequence was determined by hybridizing to targets containing a single mismatched base and determining the strength of the resulting complexes (delta G3 ₇ ) by heat denaturation. Eight targets were synthesized which were complementary to round 8 and linear oligonucleotide 9, with the exception that there was a single centrally located variable base (X or YA, G, C, T). Four targets had a variable X base, which was matched with the opposite T in the circle, resulting in a TXT triad. For the remaining four targets, the variable Y base was matched to the two opposing Cs in the circle, resulting in a CYC triad. To compare this circle with the complex, linear oligonucleotide 9 was used, which resulted in a duplex where a central TX pair was formed in the first four experiments or a CY pair in the remaining four.

· «·· * · complex (X, Y = A, T, G, C) ____ experiment number

3 '- T T C T T T T C T T T C 5 '- THE THE G THE X THE THE G THE THE THE G 1-4

C T T c T T T T c T T T c C THE C THE THE G THE X THE THE G THE THE THE G C 5-8 C T T C T T T T C T T T C C THE

C

5'-A A G A A A A Y A A A G

9-12

C T T c T T T T c T T T c C THE C THE THE G THE THE THE THE Y THE THE THE G C 13-16 C T T C T T T T c T T T C C THE

The complex 16 is denatured by heat with 10 mM MgCl 2> 100 mM

NaCl and 10 mM Tris-HCl (pH 7.0), and the target and circular or linear oligonucleotide concentrations were 3 µM. The experiments were repeated and the results were averaged. The melting of the oligonucleotide-target complex was measured at 260 nm. The temperature versus absorbance curves thus generated showed a single transition from the bound to the free oligonucleotide «· ··· ♦ · · · ·. The free energy of the connection was obtained by fitting the data with a two-step curve fitting method. The results were verified in two cases by measuring the association energies by the van't Hoff method, and a good agreement was found between the two methods. Selectivity was defined as the difference in free energy (deltaG) of complex formation between the matching and non-matching oligomers.

In III. The following table shows the results of the mismatched experiments. Experiments 1-4 show the effect of TX target mismatch on a DNA duplex. As expected, true fit (X = A) gives the most preferred complex (-delta G ₃₇ = 10.3 kcal / mol); non-fits (X = G, C, T) cause a loss in binding energy of 3.2 - 4.4 kcal / mol, which is in good agreement with published non-fitting experiments. The

5-8 experiments, comparing the effect of T-X-T mismatch on the binding energy of a circular complex. Again, true fitting (X = A) yields the most preferred triple-stranded complexes (-Delta G37 = 16.4 kcal / mol). However, target mismatches (X = G, T, C,) resulted in significantly greater binding energy loss (6.2-7.6 kcal / mol) for the circular oligonucleotide than for the linear oligonucleotide.

Similarly, experiments 9-12 provide the effect of C-Y mismatch on the duplex duplex. The Matched Base (Y = G)

-10.3 kcal / mol gave duplex coupling free energy. Non-fits (Y = A, T, C) in the binding energy 5.2 73

V · 4 ·· <····

4 «4 · V« • · <· · · · · · · · · · · · · · · · · · · · · · · ·

They caused a loss of 5.8 kcal / mol, which was also in good agreement with the published data. In contrast, a CYC mismatch has a much greater effect on a triple-stranded complex (experiments 13-16): the fit (Y = G) gives -16.4 kcal / mol binding energy, and the mismatch (Y = A, T, C) much less stable 7.1 -

7.5 kcal / mol.

Thus, in all cases examined, circular ligands exhibited greater selectivity for their correctly aligned sequences than standard linear oligomers. The selectivity advantage ranges from 1.3 to 2.2 kcal / mol in the C-Y-C series and 3.0 to 3.4 kcal / mol in the T-X-T series. These are very significant differences if we consider that they are due to a change in only one base; in the T-X-T series, the circular oligonucleotide is nearly twice as selective as the linear oligonucleotide. This selectivity difference corresponds to an order of magnitude 1-2 in the binding constant at 37 ° C.

There are two factors that can account for this high selectivity. First, since the two domains of the circular oligonucleotide bind to the central target strand, the circular oligonucleotide, when six, double-checks the sequence for correct alignment. Second, the protonization of cytosine within the C + G-C triad may also be a factor contributing to selectivity. This protonization seems to be preferred only when base triad formation occurs. creating a guanine that can share a positive charge; evidence indicates that the pKa of the cytosine within the base triad is 2-3 units within the basal triad, · · · · · · · · · higher than free deoxycytosine. The addition of this positive charge can reduce the repulsive effect of the negative charge due to the high density of phosphates in the complex and thus increase the binding stability.

Thus, circular oligonucleotides, as shown, may have greater binding affinity and greater selectivity than can be achieved alone in the Watson-Crick duplexes.

III. TABLE experiments variable Tm'C -delta G37 select number base (kcal / mol) controversial

1 X = A 43.8 10.3 2 X = G 33.8 7.1 3.2 duplex 3 X = C 28.3 5.9 4.4 4 X = T 31.1 6.4 3.9 5 X = A 62.3 16.4

6 X = G 44.2 10.2 6.2 circular complex 7 X = C 39.8 8.8 7.6 8th X = T 40.8 9.1 7.3 9 Y = A 26.2 5.1 5.2 10 Y = G 43.8 10.3 - duplex 11 Y = C 22.2 4.5 5.8 TABLE III Continue 12 Y = T 27.0 5.0 5.3 13 Y = A 39.9 9.0 7.4 14 Y = G 62.3 16.4 circular complex 15 Y = C 41.3 9.3 7.1 16 Y = T 39.6 8.9 7.5

Example 4

Factors influencing complex formation

1) The effect of the solutions is illustrated below We investigated the effect of NaCl, Mg ²⁺ , spermine and pH: target complex. Circuits containing cytosine in the binding domain are sensitive to pH and show greater stability at lower pH values. However, these and other target complexes are also quite stable at physiological pH 7.0-7.4 (Figure 5). Complexes are also sensitive to salt concentration relative to duplexes, but small amounts of Mg ²⁺ or spermine significantly increase the stability of the complexes. For example, at a concentration of 1 mM Mg ²⁺ and a pH of 7.0 without the addition of a salt, a stable 7: 5 round: target complex was formed with a Tm of 58 ° C. When a solution of 20 µM spermine was used without additional salt, the 7: 5 complex was again stable with a Tm of 56 ° C. Both Mg ^2-1 · and spermine are present in mammals at least at such concentrations, so that the target complexes will be stable under physiological conditions.

2) The size of the loop is given below

In order to determine the optimal number of nucleotides in a loop loop domain, complex formation between a single target and different loop size loops was observed. At age similar to that of Primer 1, linear oligonucleotides were synthesized 2, 3,

Using any sequence of C and A residues with alternating 4, 5, 6, and 10 base loops. Each of the curves was designed to attach to the A ^ ₂ template (e.g., Target 4, (SEQ ID NO: 8)). Tm values rendelekző 4, 5, 6 and 10 base loops loops showed that a loop of 5 nt was size optimal circle upon binding to either the ₁₂ therapy, promoting or ₁₂ binding site comprising more than 36 sequence (see Figures 6A figure).

2) The length of the binding domain is given below

The effect of the circular oligonucleotide binding domain on the melting temperature of the round: target complex was compared to the melting of duplexes of the same length. Circles with binding domains of different sizes were created and complexed with single-stranded dA _n targets, where n is identical to 4, 8, 12 and 18 nucleotides. Figure 6B shows that significantly higher Tm values for the round: target complexes were observed for Watson-Crick duplexes of the same length as the binding domains (0.1 M NaCl, pH 7). For example, a 12-base circular complex melts at about the same temperature as a 24-base duplex. The 4-base circular complex melts at 34 ° C while the corresponding Watson-Crick duplex has a Tm of less than 0 ° C.

4) Methylation is described below

It has been known for some time that naturally occurring m ^ c

- Methylation of cytosine at position C-5, which produces 78 bases, increases the Tm of duplex DNA in which it occurs relative to non-methylated sequences (Zmudzka et al., 1969, Biochemistry, 8: 3049). To investigate whether the addition of this methyl group stabilizes the circle: target complexes, two analogs of circle 7 (represented by SEQ ID NO: 6) were synthesized. In one round, 6 Cs in the binding domain were methylated, leaving the loop unmethylated (Meg). In the second round, all 12 C were methylated (Me · ^). The melting point of the complexes of this methylated ring with the purpose of 5 was measured. The Meg complex has a Tm of 71.1 ° C (relative to the 61.8 ° C of the unmethylated ring) and a Me ₁₂ Tm of 72.4 ° C. Thus, the use of natural m ^ C base instead of C significantly increased the stability and in one case resulted in a 12-base complex that melted at 10.6 C higher than unmethylated and 28.6 C higher than the corresponding non-methyl base. methylated Watson-Crick duplex.

Example 5

Substitution of nucleotide loop domains by non-nucleotide loop domains

The loop domains of circular oligonucleotides were exchanged for polyethylene or oligoethylene glycol chains of various lengths and the effect of such synthetic loops on circular oligonucleotide binding and nuclease resistance was investigated.

Following are the methods used to do this

Circular oligonucleotides having tetra-, penta-, or hexaethylene glycol-chain loop domains were synthesized. In each case, ethylene glycol chains were synthesized using automated DNA synthetic methods using the method of Durand et al. (1990, Nucleic Acids Res., 18: 6353-6359). Briefly, a phosphoramidite is placed on a hydroxyl group at one end of the ethylene glycol chain and a dimethoxytrityl (DMT) unit is placed on the other terminal ethylene glycol hydroxyl group. This derived ethylene glycol chain was added to the growing linear oligonucleotide at the appropriate step of automated DNA synthesis. The circularization step was carried out according to the procedures described in Example 1. A linear oligonucleotide having a tetraethylene loop domain did not efficiently encircle at age. This result shows that a tetraethylene loop may be too short for optimal binding to a target. Two types of linear oligonucleotides were used as target binding domains for circular oligonucleotides: Target I was a 12-base oligonucleotide lacking non-target nucleotides and Target II was a 36-base oligonucleotide with a 12 base target within the oligonucleotide. The target sequence used is 5'-AAGAAAAGAAAG-3 '(SEQ ID NO: 9) and 5'-AAAAAAAAAAAA-3' (SEQ ID NO: 8), which defines a poly (dA) ^ 2 winter sequence.

Melting points (Tm) of circular oligonucleotides having a polyethylene loop were observed at pH 7.0 (10 mM Tris-Cl) in 10 mM MgCl ₂ and 100 mM NaCl. All linear targets and all circular oligonucleotides were present at a concentration of 3 µM.

The results of the study are described below

The circular oligonucleotide having a CACAC nucleotide loop sequence and a poly (dA) ₁₂ sequence for both the P and AP domains had a Tm of 57.8 C when bound to a poly (dA) ₁₂ target sequence. A circular oligonucleotide having the same P and AP domain sequences but hexaethylene glycol loop domains had a Tm of 51.4 C when bound to the same target.

Comparison of Tm values for circular oligonucleotides with pentaethylene glycol (PEG) and hexaethylene glycol (HEG) loop domains is shown in Table IV. This is shown in the table.

«

ARC. TABLE complex

Purpose Purpose

I Tm II Tm

PEG

PEG

HEG

HEG

HEG

HEG

* · · ·· • · «♦ ·

The Tm observed for a circular oligonucleotide having an HEG loop is about. 4.5 C higher than a circular oligonucleotide having a PEG loop. Consequently, circular oligonucleotides having hexaethylene glycol loop domains have greater stability than circular oligonucleotides having tetra- or pentaethylene glycol loops.

The following is the nuclease resistance

Circular oligonucleotides were tested for nuclease resistance in the unbound and bound state to the target oligonucleotide. All circular oligonucleotides, whether bound or not, showed total resistance to exonucleases. Endonuclease sensitivity was determined using S1 nuclease as recommended by the manufacturers.

Comparison of S1 nuclease resistance of bound and unbound circular oligonucleotides is provided by V.

This is shown in the table.

TABLE V oligonucleotide cleavage

Time for 50% Sl

HEG

HEG

HEG

HEG> 24 hours

minute

minute

• · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ··· ·

These data indicate that unbound circular oligonucleotides are sensitive to S1 nuclease. However, when bound to a target, a circular oligonucleotide having a polyethylene loop domain is much more resistant to Si1 nuclease, at least 36 times more resistant than a circular oligonucleotide having a nucleotide loop domain.

The nuclease resistance of circular and linear oligonucleotides was also compared when the oligonucleotides were incubated in human plasma for different periods of time. Circular oligonucleotide 7 and its precursor and linear oligonucleotide 2 were incubated at a concentration of 50 µM in plasma at 37 ° C. At different times, the same amounts were removed and the cleavage products were separated by gel electrophoresis. Nuclease resistance was tested by observing whether degradation products were evident on the gels.

Linear oligonucleotide 2 had a half-life of 20 minutes when incubated in human plasma. In contrast, circular oligonucleotide 7 did not undergo measurable nuclease digestion during 48 hours of incubation. Accordingly, a circular oligonucleotide has a half-life greater than 48 hours in human plasma, 140 times longer than a linear oligonucleotide having the same sequence.

Example 6

Circular oligonucleotides may selectively bind to RNA.

The experiments described in this example indicate that, unlike linear oligonucleotides, circular oligonucleotides may prefer to bind to an RNA rather than a DNA target.

Two linear deoxyoligonucleotides were generated as targets, the T (SEQ ID NO: 11) target and one dU (SEQ ID NO: 12) target:

Target T: 5'-AAGAATAGAAAG-3 '; and dU Purpose: 5'-A A G A A U A G A A A G-3 '.

A circular oligonucleotide of SEQ ID NO: 14 was also prepared:

For comparison, a linear oligonucleotide complementary to the T and dU targets was also synthesized (e.g., a linear oligonucleotide having the sequence SEQ ID NO: 13). ):

5 '

The melting temperatures (Tm) of circular vs. linear oligonucleotides bound to each target were monitored and shown in Figure VI. This is shown in the table.

VI. SPREADSHEET

Tm values for oligonucleotides

Objectives Linear Circular

Target T 42.9 C 41.1 Target C dU 40.9 C 42.9

C______________________________________________________________________

The linear oligonucleotide binds to the T target much more strongly than to the dU target, to an extent that is significantly greater than the experimental error limits. This difference in Tm corresponds to a difference in free binding energy of 1.7 kcal / mol.

However, in contrast to the linear oligonucleotide, the circular oligonucleotide binds much more strongly to the U target. Therefore, the circular oligonucleotide may show a preference for an RNA target over an appropriate DNA target.

In addition, the increase in binding energy of the circular oligonucleotide to the RNA target corresponds to a free energy difference of 0.8 kcal / mol, indicating that an RNA target at 37 ° C would be preferred. 3: 1 ratio to a suitable DNA target.

Example 7

Fiber displacement of circular oligonucleotides

Circular oligonucleotide 6 (Figure 3) binds to a dA ₁₂ target with 9 kcal / mol more stability than a linear dTj1 oligonucleotide (Example 2). This increase in stability indicates that a circular oligonucleotide: target complex thermodynamically favors a linear oligonucleotide: target complex. Furthermore, a circular oligonucleotide can actually accelerate (or catalyze) the dissociation of a duplex DNA target sequence to form a complex with one strand of the duplex.

To investigate whether a circular oligonucleotide can readily dissociate duplex DNA and displace a strand of the duplex DNA target, we have observed the complementarity of the strand movement kinetics of a duplex DNA target · ·· »

In the presence of 88 linear or circular oligonucleotides.

A DNA duplex target having a fluorescent moiety on one strand and a tetramethylrhodamine moiety on the other end was prepared (Cardullo et al., 1988, Proc. Natl. Acad. Sci. USA 85: 8790; Cooper et al. , 1990 Biochemistry .29 .: 9261). The structure of the duplex target (SEQ ID NO: 15) was as follows:

5'-Fluorescein-A AAAAAAAAA

3'-rhodamine-T TTTTTTTTT.

The Tm value of this labeled duplex target was normal, so the fluorescein substitutions had no significant effect on the association kinetics. Furthermore, the emission maximum of the fluorescein-dA-L ₂ strand was at 523 nm, whereas the rhodamine-dT | _The emission maximum of ₂ fibers was at 590 nm, allowing the association kinetics of the two fibers to be investigated separately.

The fiber displacement reaction was performed at 10 ° C in a 1 cm fluorescence cuvette. Reaction conditions: 100 mM NaCl, 10 mM MgCl 2 and 10 mM Tris-HCl, pH 7.0, reaction volume 3 ml. The labeled duplex was allowed to equilibrate at 10 ° C for at least one hour before the addition of a 40-fold predominant linear or circular oligonucleotide (final concentration 0.01 µM). A Spex Flurolog F 111A fluorescence device was used with a 5 mm slit width. The excitation wavelength was 450 nm and the measured emission wavelength was 523 nm. The results were independent of both the excitation and the measured emission wavelengths. The reaction was followed for at least 5 half-lives.

The rhodamine-dT-dA ^ ^ ₂ ^f luoreszcein -höz administration caused a decrease in fluorescein fluorescence and an increase in rodaminéban. Such effects are due to energy transfer between fluorescent units (Cardullo et al.).

The association rate constant of the two fluorescently labeled fibers was determined by mixing the fibers under pseudorandom conditions and determining the extent of reduction in fluorescent emission. The observed association constant at 10 ° C

3.2 X 10 ⁶ sec ^-1 was in good agreement with the published association rate of DNA oligonucleotides (Nelson et al., 1982, Biochemistry .21: 5289; Turner et al., 1990 in: Nucleic Acids (Vol. C), W. Saenger, Ed. Springer-Verlag, Berlin: 201-227).

A single linear fiber (SEQ IS NO: 8) or a SEQ ID

A circular oligonucleotide having the NO: 5 sequence (e.g., a

Circular Oligonucleotide 6) was used to compare predominant amounts of unlabeled linear or circular oligonucleotides in a duplex DNA to the fluorescent labeled duplex DNA. The increase in fluorescent emission was then measured at a temperature significantly below the Tm of the duplex target, which is a measure of the dissociation of the duplex target strand.

Figure 8 shows a typical kinetic assay for the dissociation of a duplex target with the presence of an unlabeled dA-? ₂ (dotted line) or 40x predominant circular oligonucleotide at 10 ° C. As described, dissociation of the duplex target by a circular oligonucleotide is significantly faster than dissociation by a linear oligonucleotide. The first ratio constant of the dissociation by the linear oligonucleotide is 2.0 X 10 ^-4 sec ^-1 , whereas the constant by the circular oligonucleotide is constant.

2.3 X 10 was ² sec ^-1 , almost 2 orders of magnitude faster. This difference is even more pronounced when calculating the half-life of the target in the presence of linear vs. circular oligonucleotides. At 10 C, the dissociation half-life of the duplex is 58 minutes in the presence of a linear oligonucleotide but only 30 seconds in the presence of a circular oligonucleotide.

Unlike the rate of reaction between the linear oligonucleotide and the duplex, the rate of reaction between the circular oligonucleotide and the duplex is dependent on the concentration of circular oligonucleotide added at low concentration and exhibits a Michaelis-Menten type saturation property at higher concentrations (Figure 9).

The dissociation rate of the labeled duplex at 10 ° C can be calculated from the duplex association rate constant and the delta Gq values. This rate constant, 8.5 X 10 ^-1 θ sec ^-1 , is equivalent to that derived from estimated thermodynamic parameters for duplex complexes (Breslauer et al., 1986, Proc. Natl.Acad. Sci. USA 83: 3746), although it is the rate is significantly less than the rate constant of the strand displacement by a linear oligonucleotide. An increase in the dissociation of the duplex in other cases has been observed with the addition of linear oligonucleotide r ····· · · · (Chamberlin et al., 1965, J.Mol. Bioi., 12: 410). Comparison of the rate of the circular oligonucleotide-catalyzed reaction and of the non-catalyzed duplex dissociation revealed an increase of about 10 ⁷ -fold (Sigler et al., 1962, J.Mol. Biol. 5: 709).

The 1 / [circular oligonucleotide] vs.l / k _{to w} ff. the double reciprocal plot is linear and K _cat 0.024 +, - 0.005 sec ^-1 and KM 2.2 x 10 ⁷ M. The kkat is 100-fold greater than the observed rate constants of the duplex reaction with either dA ₁₂ or dT ₁₂ single fibers.

The observed saturation behavior (Figure 9) suggests that a complex is formed between the circle and the double-stranded target. Using the above Kjyj value, and assuming that the k _cat << _lf where k_ K_F at 10 C, the complex dissociation rate constant of the association free energy of -8.6 kcal / mol. This value is similar to the estimated -9 kcal / mol for the P domain in a 12-base triple helix containing TAT base triads as reported by Pilch et al. al. (1990, Nucleic Acids Sl. 18: 5743).

The following is a list of sequences.

·· «. «·· *>

• · ··

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(ii) TITLE OF THE INVENTION: monofilament; CIRCULAR OLIGONS -

LEOTIDS (iii) NUMBER OF SEQUENCES: 15 (iv) ENTRY TITLE:

(A) ADDRESSED: Scully, Scott, Murphy & Presser (B) STREET: 400 Garden City Plaza (C) CITY: Garden City (D) STATE: New York (E) COUNTRY: USA (F) DIRECTORY: 11530 (V) COMPUTER READABLE FORM:

(A) MATERIAL TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patent in Release # 1.0, Version (vi) APPLICATION DETAILS:

(A) APPLICATION NUMBER: US (B) EDUCATION DATE:

CLASSIFICATION (C):

(Vili) LAWYER / OFFICE INFORMATION:

(A) NAME: McNulty, William. E.

(B) REGISTRATION NUMBER: 22.606 (C) REFERENCE / LIST NUMBER: 8085Z (ix) TELECOMMUNICATIONS INFORMATION:

(A) PHONE: (516) 742-4343 (B) TELEFAX: (516) 742-4366 (2) SEQ ID NO: 1 INFORMATION:

(i) SEQUENCE FEATURES:

(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: single stranded (D) TOPOLOGY: circular (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 1 CTCCCCGCCC TCNNNNCTC CCACCCCTCN NNN (2) information about:

(i) SEQUENCE FEATURES:

(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: single-stranded (D) TOPOLOGY: circular (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 TCTTTTTTCT TTTCNNNNC TTTTCTTTTT TCTN information about:

(i) SEQUENCE FEATURES:

(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: single-stranded (D) TOPOLOGY: circular (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3 TTTCYTCGTT CGTCNNNNNC TACTTACTNC TACTTACTNC information about:

(i) SEQUENCE FEATURES:

(A) LENGTH: 40 base pairs (B) TYPE: Nucleic Acid (C) Fiber Type: Single-stranded (D) TOPOLOGY: Circular (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 TCCTTCTTCY CCTCTNNNNN TCTCCGTNN information about:

(i) SEQUENCE FEATURES:

(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: single-stranded (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5

TTTTTTCACA CTTTTTTTTT TTTCACACTT TTTT (2) SEQ ID NO: 6 INFORMATION:

• · · · · «· · · · · · · · · · · · · · · · · · · · · · · ···

- 96 (i) SEQUENCE FEATURES:

(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: single-stranded (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6

TCTTTCCACA CCTTTCTTTT CTTCACACTT CTTT 34

(2) SEQ ID NO: 7 INFORMATION:

(i) SEQUENCE FEATURES:

(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: single-stranded (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7

TTTCTTCACA CTTCTTTTCT TTCCACACCT TTCT (2) SEQ ID NO: 8 INFORMATION:

(í) SEQUENCE FEATURES:

(A) LENGTH: 12 base pairs (B) TYPE: Nucleic acid (C) FIBER TYPE: single-stranded (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 AAAAAAAAAAA AA (2) SEQ ID NO: 9 INFO:

(i) SEQUENCE FEATURES:

(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: single-stranded (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9

AAGAAAAGAA AG (2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE FEATURES:

(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: single-stranded (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10

CTTTCTTTTC TT (2) SEQ ID NO: 11 INFORMATION:

(i) SEQUENCE FEATURES:

(THE) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) Fiber type: single stranded (D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11

AAGAATAGAA AG (2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE FEATURES:

(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: single-stranded (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12

AAGAAUAGAA AG (2) SEQ ID NO: 13 INFORMATION:

(i) SEQUENCE FEATURES:

(THE) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) Fiber type: single stranded (D) TOPOLOGY: linear

- 100 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13

CTTTCTATTC TT (2) SEQ ID NO: 14 INFORMATION:

(i) SEQUENCE FEATURES:

(A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: single-stranded (D) TOPOLOGY: both (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14

TTCTTCTCTT TCCACACCTT TCTATTCTTC ACAC (2) SEQ ID NO: 15 INFORMATION:

(i) SEQUENCE FEATURES:

(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) FIBER TYPE: double-stranded (D) TOPOLOGY: linear

- 101 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 15

AAAAAAAAAA AA

Claims (64)

PATENT CLAIMS A single-stranded circular oligonucleotide comprising at least one parallel-linked (P) domain and at least one anti-parallel-linked (AP) domain, having a loop domain between each binding domain involved in the generation of the circular oligonucleotide; each of the P and corresponding AP domains having sufficient complementarity to detectably bind to a strand of a particular nucleic acid target, wherein said P domain binds parallel to said target and said corresponding AP domain binds anti-parallel to said target. 2. The oligonucleotide of claim 1, wherein said target comprises a known nucleotide sequence from which a nucleotide sequence is determined from said target sequence for a sufficient number of positions of said P domain and said corresponding AP domain: for said P domain: if, for said purpose, a base is a guanine or a guanine analog at one position, P has a cytosine or a suitable analogue thereof at a suitable position; if, for said purpose, a base is an adenine or an adenine analog at one position, P, at a suitable position, P has thymine or uracil or a suitable analogue thereof; 103 when said base is a base thymine or a thymine analog at said position, wherein at a suitable position P has a cytosine or guanine or a suitable analogue thereof; if for that purpose a base is a cytosine or a cytosine analog at one position, then at a suitable position P has a cytosine, thymine or uracil or a suitable analogue thereof; and if, for said purpose, a base at one position is a uracil or a uracil analogue, then at a suitable position P has a cytosine, guanine, thymine, or uracil, or a suitable analogue thereof; for said AP domain: if, for said purpose, a base is a guanine or a guanine analog at one position, the AP has a cytosine or a uracil or a suitable analogue thereof at a suitable position; if for said purpose a base is an adenine or an adenine analog at one position, then the AP has a thymine or uracil or a suitable analogue thereof at a suitable position; if, for said purpose, a base thymine or a thymine analog at one position, the AP has an adenine or a suitable analogue thereof at a suitable position; if for that purpose a base is a cytosine or a cytosine analog, then at a suitable position the AP is a · · 4 · 4 · · 44 ······· 4 4 · 44 ··· 104 guanine or a suitable analogue thereof; and if for that purpose a base is a uracil or a uracil analog at one position, the AP has an adenine or a guanine or a suitable analogue thereof at a suitable position; wherein said sufficient number of positions is the number of positions which provides sufficient complementarity for the oligonucleotide to detectably bind to said target. The oligonucleotide of claim 1, wherein said P domain comprises a nucleotide sequence as determined from the nucleotide sequence of said target: if, for said purpose, a base is a guanine or a guanine analog at one position, P has a cytosine or a suitable analogue thereof at a suitable position; if, for said purpose, a base is an adenine or an adenine analog at one position, then in a suitable position P has a thymine or uracil or a suitable analogue thereof; if, for said purpose, a base is a thymine or a thymine analog at one position, then at a suitable position P has a cytosine or guanine or a suitable analogue thereof; if for that purpose a base is a cytosine or a cytosine analog, then at a suitable position P is a · · · · · · · · · · · · · · · · · · · · · · ·· - 105 cytosine, thymine or uracil or a suitable analogue thereof; if for that purpose a base is a uracil or a uracil analog at one position, then at a suitable position P has a cytosine, guanine, thymine or uracil, or a suitable analogue thereof; and further wherein said AP domain has a nucleotide sequence as determined from said sequence of said target as follows; if, for said purpose, a base is a guanine or a guanine analog at one position, the AP has a cytosine or a uracil or a suitable analogue thereof at a suitable position; if for said purpose a base is an adenine or an adenine analog at one position, then the AP has a thymine or uracil or a suitable analogue thereof at a suitable position; if, for said purpose, a base thymine or a thymine analog at one position, the AP has an adenine or a suitable analogue thereof at a suitable position; if for that purpose a base is a cytosine or a cytosine analog at one position, then the AP has a guanine or a suitable analogue thereof at a suitable position; and if, for said purpose, a base is a uracil or a uracil analog at one position, then at a suitable position the AP with an adenine or a guanine or a suitable analogue thereof 106 has. The oligonucleotide according to claim 1, 2 or 3, wherein said target, said P domain and said AP domain are each independently about one to one in length. It contains 2-200 nucleotides. 5. The oligonucleotide of claim 4, wherein said target, said P domain and said AP domain, is independently about one to one in length. It contains 6-36 nucleotides. 6. The oligonucleotide of claim 1, 2 or 3, wherein each loop domain is independently about one to one in length. They contain 2-2000 nucleotides. 7. The oligonucleotide of claim 6, wherein each of the loop domains is independently about one to one in length. They contain 3-8 nucleotides. 8. The oligonucleotide of claim 1, 2 or 3, wherein said target has a single or double strand. The oligonucleotide of claim 1, 2 or 3, wherein said target is RNA or DNA. 10. The oligonucleotide of claim 1, 2 or 3, wherein said target is a domain comprising a template of a nucleic acid. 11. The oligonucleotide of claim 1, 2 or 3, wherein said P domain and said • ·· ··· - 107 - The P domain binds to said target in a stepwise binding arrangement. The oligonucleotide according to claim 1 or 2, wherein the satisfactory complementarity is less than 100% complementarity. 13. The oligonucleotide of claim 12, wherein the satisfactory complementarity is about. 30% to 40% complementarity. 14. The oligonucleotide of claim 1, 2 or 3, wherein said oligonucleotide can be DNA or RNA. 15. The oligonucleotide of claim 2 or 3, wherein the suitable cytosine analog is 5-methylcytosine. The oligonucleotide according to claim 2 or 3, wherein the suitable uracil analog is 5-methyluracil. 17. The oligonucleotide of claim 2 or 3, wherein diaminopurine is a suitable adenine analog. 18. The oligonucleotide of claim 1, 2 or 3, wherein the nucleotides have a 2'-O-methylribose instead of ribose or deoxyribose. 19. The oligonucleotide of claim 1, 2 or 3, wherein said oligonucleotide is uptake by a cell. 108 • ♦ · · 4 · «·· ·· · 4 * · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 20. The oligonucleotide of claim 19, wherein said oligonucleotide further comprises a ligand that serves as a cellular receptor, a cholesterol group, an aryl group, a steroid group, or a polycation. 21. The oligonucleotide of claim 1, 2 or 3, wherein said oligonucleotide further comprises a drug or a drug analog. 22. The oligonucleotide of claim 1, 2 or 3, wherein said loop domains comprise non-nucleotide loop domains. 23. The oligonucleotide of claim 22, wherein said non-nucleotide loop domains consist of polyethylene glycol. The oligonucleotide of claim 23, wherein said polyethylene glycol may be pentaethylene glycol, hexaethylene glycol or heptaethylene glycol. 25. The oligonucleotide of claim 1, 2 or 3, wherein said oligonucleotide further comprises at least one methylphosphonate, phosphorothioate, phosphorodithioate, phosphotriester, siloxane, carbonate, acetamidate, thioether or phosphorus boron. 26. The oligonucleotide of claim 1, 2 or 3, wherein said oligonucleotide further comprises a reporter molecule. 27. A target for the detection or diagnosis of nucleic acid - a kit consisting of 109 parts for tizing, characterized in that: 1-3. At least one first compartment for providing a circular oligonucleotide according to any one of claims 1 to 6. A kit comprising parts for isolating a template nucleic acid, comprising at least one first compartment for providing a circular oligonucleotide according to claim 1, 2 or 3, wherein said oligonucleotide exhibits complementarity with said target within said template. 29. The kit of claim 28, wherein said template is a poly (A) ⁺ mRNA. 30. A method of regulating DNA, RNA, or protein biosynthesis, comprising: 1-3. contacting at least one of the oligonucleotides of any one of claims 1 to 9 with a nucleic acid template of said DNA or RNA or protein under conditions sufficient to bind at least one of the oligonucleotides to the target sequence contained in said template; said oligonucleotide binds to said target; blocking contributes to or permits degradation of said template and thereby regulates the biosynthesis of said DNA, RNA or protein. 31. The method of claim 30, wherein said template comprises a double-stranded nucleic acid target and said conditions are effective for denaturing said target by displacement of said target thereby binding. - 110 - they allow. 32. The method of claim 30, wherein said biosynthesis comprises at least one of DNA replication, DNA reverse transcription, RNA transcription, RNA cleavage, RNA polyadenylation, RNA translocation, and protein translation. 33. The method of claim 32, wherein said template for replication of said DNA can be an RNA template or a DNA template. 34. The method of claim 33, wherein said target of said oligonucleotide for regulating said DNA replication may be an origin of replication or a primary binding site. 35. The method of claim 32, wherein said target of said oligonucleotide for regulating reverse transcription of said DNA is a primary binding site, a retroviral site, or a mRNA site. 36. The method of claim 32, wherein said target of said oligonucleotide for regulating transcription of said RNA is a promoter, a repressor binding site, an operator, an enhancer, a transcriptional regulatory element, or a site in an mRNA coding region. . 37. The method of claim 32, wherein said target of said oligonucleotide for controlling RNA cleavage is a 5 'cleavage point, an intron cleavage point, or a 3' cleavage point. «* ·· <· - 111 38. The method of claim 32, wherein said target of said oligonucleotide for controlling polyadenylation of said RNA may be a polyadenylation site. 39. The method of claim 32, wherein said target of said oligonucleotide for controlling translocation of RNA is a poly (A) tail. 40. The method of claim 32, wherein said template for translation of said protein can be an mRNA template. 41. The method of claim 40, wherein said target of said template is a ribosome binding site, a 5'mRNA cap or a site in a protein coding region. 42. The method of claim 30, wherein said template is a viral DNA or RNA template. 43. The method of claim 42, wherein said oligonucleotide has a nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4. 44. A method of moving a strand in a double-stranded nucleic acid target comprising: associating said target with a method according to any one of claims 1-3. A circular oligonucleotide according to any one of claims 1 to 6 for a period of time, and under conditions effective to denature said target and said circular oligonucleotide. 112 • 4 »·· 444 * · * · ·« 6 • ··· »44 ··· f · 4 ♦ · 44 * «·« «» »* oligonucleotide binding. The method of claim 44, wherein said conditions are effective for denaturing said target, characterized in that said circular oligonucleotide and said target are present in an amount of ca. Includes 1-100 ratios. 46. The method of claim 45, wherein said time is effective for denaturing said target, wherein the time interval is from about 1 minute to about 1 minute. Up to 16 hours. 47. The method of claim 44, wherein said double-stranded nucleic acid target comprises a viral, a bacterial, a fungus, or a mammalian nucleic acid. 48. The method of claim 47, wherein said double-stranded nucleic acid target is an origin of replication, a promoter, a repressor binding site, an operator, an enhancer, a transcriptional regulatory element, or a site in an mRNA coding region. 49. The method of claim 44, wherein said double-stranded nucleic acid target is present in a sample of pure or non-pure nucleic acid, a tissue portion, a cell smear or a chromosomal mass. 50. The method of claim 49, wherein said oligonucleotide is covalently linked to a reporter molecule. 51. A pharmaceutical composition for regulating the biosynthesis of a nucleic acid or protein, wherein the composition of any one of claims 1-3. Oligonucleotides according to any one of claims 1 to 4 At least one of 113 is contained in a biosynthesis regulating amount and a pharmaceutically acceptable carrier. 52. A method of producing single-stranded circular oligonucleotides according to claim 1, 2 or 3, comprising binding to a linear live oligonucleotide, linking the two ends of said living age and recovering said single-stranded circular oligonucleotide. 53. The method of claim 52, wherein said linear age has a 3'-phosphate. 54. The method of claim 53, wherein said two ends comprise two nucleotides corresponding to the AP nucleotides of said single-stranded circular oligonucleotide. 55. The method of claim 54, wherein said coupling is performed with BrCN, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide or N-cyanoimidazp1 ZnCl2. 56. A complex comprising the oligonucleotide of claim 1, 2 or 3 and the target being formed. 57. A method of delivering a drug to a particular cell type comprising: (a) administering to an animal a drug covalently bound to the oligonucleotide of claim 1, 2 or 3; (b) said oligonucleotide is by said cell - 114 ·· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · and (c) thus delivering said drug to said specific cell type. 58. A method for detecting a target nucleic acid comprising: contacting any of the circular oligonucleotides according to claim 1, 2 or 3 for testing a sample of said amino acid content for a period of time and under conditions sufficient to form the oligonucleotide-target complex; and detecting said complex. 59. The method of claim 58, wherein said nucleic acid comprises a double-stranded nucleic acid target and said conditions are effective for denaturing said target strand displacement and thereby allowing said oligonucleotide to form said oligonucleotide-target complex. 60. The method of claim 58, wherein said sample comprises a sample of pure or non-pure nucleic acid, a piece of tissue, a cell smear, or a chromosomal mass. 61. The method of claim 58, wherein said conditions are effective for denaturing said target, characterized in that said circular oligonucleotide and said target are present in an amount of ca. Includes 1-100 ratios. 115 4 «· ··· • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 62. The method of claim 58, wherein said time is effective for denaturing said target, wherein the time interval is from about 1 minute to about 1 minute. Up to 16 hours. 63. The method of claim 58, wherein said complex is detected by fluorescence energy transfer assay. 64. The oligonucleotide of claim 12, wherein the satisfactory complementarity is at least 50%.