WO2015180887A1

WO2015180887A1 - Device and method for detecting and quantifying single-stranded target nucleic acids

Info

Publication number: WO2015180887A1
Application number: PCT/EP2015/058297
Authority: WO
Inventors: Stefan Hofmann; Walter Gumbrecht; Yiwei Huang
Original assignee: Siemens Aktiengesellschaft
Priority date: 2014-05-27
Filing date: 2015-04-16
Publication date: 2015-12-03
Also published as: DE102014210092A1

Abstract

The invention relates to a device and a method for detecting and quantifying single-stranded target nucleic acids. The device comprises at least one solid carrier on which at least one double-stranded capture oligonucleotide and at least one double-stranded reporter oligonucleotide are immobilised. The capture oligonucleotide comprises at least one first oligonucleotide sequence that is partially complementary to a target nucleic acid, and the reporter oligonucleotide comprises at least one second oligonucleotide sequence that is partially complementary to the target nucleic acids. The device also comprises a reading unit for reading a marker of the reporter oligonucleotide on the carrier.

Description

description

Apparatus and method for detecting and quantifying single-stranded target nucleic acids

The invention relates to an apparatus and a method for the detection and quantification of single-stranded target nucleic acids. The expression of genes in cells is tightly controlled and is re ^¬ guliert through various molecular processes. In addition to the complex regulation of transcription of genes, mechanisms at the post-transcriptional level, that is, at the level of mRNA, also play an important role. In particular, short non-coding nucleic acid

Engage molecules in gene expression by specifically anneal to complementary sequences of mRNA molecules, and thereby change the rate of translation of the mRNA into the ent ^¬ speaking protein. The short nucleic acid molecules are often called. MicroRNAs (short "miRNAs"), which have increasingly attracted the interest of molekularbio ^¬ logical and medical research in recent years. It was shown that selected miRNAs directly related to the emergence certain cancers such as breast cancer, lung cancer or leukemia are. It knows ^¬ sen cancer cells an increased or decreased number spe ^¬ zifischer miRNAs on. Even with immunological diseases, such as rheumatoid arthritis, certain miRNAs occur in altered concen ^¬ tions. It is assumed that in humans alone, there are over 1000 different miRNA molecules.

Due to the growing importance and variety of miRNAs, methods for their specific identification and quantification are needed. Various methods for the detection of miRNAs are known, for example the sequencing of the miRNAs or the amplification of miRNAs by means of real-time PCR. The US 6,322,971 describes a hybridization-based Ver ^¬ go for the detection of nucleic acid. In this case, the assigning to ^¬ nucleic acid with a second, labeled nucleic ^¬ ynoic acid covalently linked after the two nucleic acids are stored in an immobilized strand oligonucleotide to ^¬ and missing nucleotides between the nachzu ^¬ facing and the labeled nucleic acid by a DNA - Polymerase have been filled. Unbound labeled nucleic ^¬ leinsäuren be removed by increasing the temperature.

No. 6,344,316 describes a method for detecting nucleic acids, in which an immobilized capture oligonucleotide is covalently linked to a second, labeled oligonucleotide after the oligonucleotides have attached to the nucleic acid to be detected as a complementary strand. After removal of the nucleic acid to be detected by washing at high temperature, the covalently bound label remains. From the prior art, various problems arise. For example, the specificity and sensitivity of miRNA detection are inadequate. Among other things, in hybridization-based methods, the low specificity is that no complete hybridization of the miRNA is required for the identification process. Because of the clotting ^¬ gen sensitivity of the method, the miRNAs often have to be amplified before their detection. This can lead to the incorporation of defective nucleotides, so that the specificity of miRNA detection is further limited. The procedures must also be performed at low temperatures because the short length of miRNA hybrids is associated with low melting temperatures. Thus, the miRNA detection is not particularly robust yet quantitatively ^¬ cycled. In addition, many methods require pre-analytical labeling of miRNA, which entails the risk of inaccurate results and artifacts. The invention is therefore based on the object to provide a Vorrich ^¬ tion and a method for the detection of single-stranded nucleic acids, which solves the problems mentioned.

The device according to the invention for detecting at least one single-stranded target nucleic acid in a sample comprises at least one solid support on which at least one double-stranded capture oligonucleotide is immobilized. The capture oligonucleotide comprises a first catcher Oligonuk ^¬ leotid partial strand and a second oligonucleotide strand portion scavenger. The first end of the first capture oligonucleotide subunit is tightly attached to the support. The second capture oligonucleotide substrand has a first capture oligonucleotide overhang on the side away from the support.

Furthermore, the device according to the invention comprises a double-stranded reporter oligonucleotide having a first and a second reporter oligonucleotide partial strand. A first end of the second reporter oligonucleotide substrand comprises a first single-stranded reporter oligonucleotide overhang. The first capture oligonucleotide overhang comprises at least a first oligonucleotide sequence that is complementary to a first target nucleic acid segment. The first re ^¬ Porter oligonucleotide overhang comprises at least a second oligonucleotide sequence which is complementary to a second target nucleic acid portion. The first catcher oligonucleotide overhang thus hybridizes to the first target nucleic acid portion and the first reporter oligo ^¬ nucleotide overhang hybridizes to the second target nucleic acid portion.

Furthermore, the inventive apparatus comprises a reading unit for reading from ^¬ a mark of the first Reporting ter-oligonucleotide strand part on the support. The inventive method for the detection and quantification ^¬ tion of at least one single-stranded target nucleic acid comprises several steps: providing at least one FES th carrier on which a double-stranded oligonucleotide is immobilized at least scavenger. The capture oligonucleotide comprises a first capture oligonucleotide partial strand and a second capture oligonucleotide partial strand. The first end of the first capture oligonucleotide substrand is attached to the support. The second scavenger oligonucleotide substrand has a first scavenger oligonucleotide overhang on the side away from the carrier.

A further step is contacting the support with at least one single-stranded target nucleic acid and at least one double-stranded reporter oligonucleotide in a first hybridization condition, wherein the reporter oligonucleotide comprises a first and a second reporter oligonucleotide sub-strand. A first end of the second reporter oligonucleotide substrand comprises a first single-stranded reporter oligonucleotide overhang.

The first capture oligonucleotide overhang comprises at least a first oligonucleotide sequence that is complementary to a first target nucleic acid portion. Furthermore, in order ^¬ the first reporter oligonucleotide overhang summarizes at least a second oligonucleotide sequence which is complementary to a second target nucleic acid portion such that the first capture oligonucleotide overhang with the first target nucleic acid portion and the first reporter - hybridizing Oligonukleo ^¬ tid 'overhang with the second target nucleic acids section.

The method further comprises the step of ligating the target nucleic acid to the first scavenger oligonucleotide substrand and to the first reporter oligonucleotide substrand by means of a ligase. Furthermore, it comprises a first washing of the carrier under stringent washing conditions such that the Carrier of unligated nucleic acids is purified. Another step is reading out a label of the first reporter oligonucleotide partial strand on the support. The term target nucleic acid refers to a single nucleic acid having a length of in particular about 17 to 50 nucleotides et ^¬ wa. The nucleic acid may be an RNA or a DNA. In particular, the nucleic acids are naturally occurring nucleic acids as well as chemically produced or recombinantly produced nucleic acids.

The term "hybridize" denotes the attachment of a single-stranded RNA or DNA to an at least partially complementary single-stranded RNA or DNA with the formation of hydrogen bonds between the respective complementary bases, so that base pairings form in the mutually complementary section between the two nucleic acid molecules . the term "hybridization condition" refers to at least a parameter or combination of parameters is performed at the / at least one of the steps of hybridisation Ver ^¬ driving. The hybridization condition is a condition in which the capture oligonucleotide, the target nucleic acid and / or the reporter oligonucleotide anneal to the respective complementary portions of the opposite strand oligonucleotide to form base pairings. The parameters preferably include a temperature, a salt concentration, an ionic strength, a pH and / or an addition of reagents such as formamide.

By means of the device according to the invention and the method according to the invention, the specificity of the analysis of the target nucleic acid is markedly increased. The target nucleic acid is completely bound to the reporter and capture oligonucleotide. Thus, each base is analyzed without omitting defined ^{sections of} the target nucleic acid. Becomes advantageous This improves the discrimination of target nucleic acids that differ in marginal nucleotides.

Furthermore, the sensitivity is advantageously increased. After the step of hybridization of the target nucleic acid to the Dop ^¬ pelstrang the capture oligonucleotide, or the reporter oligonucleotide, and after the subsequent ligation of the target nucleic acid with the capture oligonucleotide and the reporter Oligonukleotid the target Nucleic acid both firmly attached to the carrier, as well as firmly attached to the label. The washing step thus very specifically removes only unbound or weakly bound reporter oligonucleotides or non-target nucleic acids from the support. Advantageously, this significantly reduces false-positive signals and thus increases the sensitivity. By increasing the sensitivity of an amplification of the target nucleic acid can be avoided prior to analysis ^¬ geous ago.

The method according to the invention is a quantitative measuring method which advantageously requires very short test times for detecting the target nucleic acid.

In an advantageous development and embodiment of the invention, the first capture oligonucleotide overhang and / or the first reporter oligonucleotide overhang comprises eight to 25 bases. The length of each overhang depends on the length of the target nucleic acid. The target nucleic acid rela ^¬ hung as their complementary sequence is divided between the reporter and catcher oligonucleotide overhang. This division is typically such that each nucleotide of the target nucleic acid can be hybridized to a reporter or capture overhang. However, it is also conceivable that a nucleotide sequence or a single base of the target nucleic acid is bound neither to the reporter oligonucleotide nor to the capture oligonucleotide. This is particularly advantageous for the detection of single-nucleotide polymorphisms. In a further advantageous embodiment and development of the invention, the first end of the first capture oligonucleotide partial strand comprises six thymine bases. The capture oligonucleotide is advantageous with at least one

Thymine base bound to the carrier.

In a further advantageous embodiment and development of the invention, the first reporter oligonucleotide partial strand has the label. Particularly preferably, the first reporter oligonucleotide partial strand has the label at its distal end to the carrier or to the target nucleic acid. The label is then tightly bound to the support during ligation via the target nucleic acid and the first capture oligonucleotide partial strand.

In a further advantageous embodiment and development of the invention, the ligase is a T4 DNA ligase. T4 DNA ligase is an enzyme that catalyzes the ATP-dependent ligation of two single-stranded RNA or DNA molecules. A covalent bond of the 3 'hydroxyl end of a first nucleic acid molecule to the 5' phosphoryl end of a second nucleic acid molecule is thereby produced. In particular, the T4 DNA ligase can ligate two DNA molecules with each other, two RNA molecules with one another and a DNA molecule with an RNA molecule. This is necessary because the Repoter- and the capture oligonucleotide typically grasp DNA nucleotides by ^¬, and typically RNA comprising the target nucleic acid nucleotides. However, T4 DNA ligase ligates only the 5 ^X end of a DNA with a 3 ^X end of an RNA. It does not ligate a 3 ^X end of the DNA with a 5 ^X end of the RNA.

Therefore, in a further advantageous embodiment and development of the invention, the 3 ^X end of the DNA is synthesized with at least one, preferably four, RNA nucleotides as a chimera to allow ligation. This concerns in particular the first capture oligonucleotide partial strand.

This is bound to the carrier with its 5 ^X end. The 3 ^λ end of the first capture oligonucleotide substrand is here ligated with the 5 ^X end of the target nucleic acid. But it is also conceivable that the orientation of the partial strands is bound in opposite directions on the support.

Furthermore, for the ligation of DNA and RNA with the T4 DNA ligase, the 5 ^X end of the DNA and the RNA must be phosphorylated. The target nucleic acid is in particular a miRNA. This is already naturally phosphorylated in the body. The re ^¬ Porter or capture oligonucleotide must be synthesized phosphorylated accordingly.

General summary fol ^¬ constricting for T4 DNA ligase conditions must be met for ligation: The li- to yawing 5 ^X-ends must be phosphorylated. In the case where the target nucleic acid is an RNA, the 3 ^X ends of the reporter or capture oligonucleotide to be ligated must comprise RNA nucleotides. This can be designed as an RNA strand or as a chimera, the chimera being fundamentally more stable. In a further advantageous embodiment and development of the invention, a second washing of the carrier takes place under mild washing conditions prior to ligation. In mild washing conditions, the salt concentration is increased compared to the harsh wash conditions and / or the temperature is lowered. In harsh wash conditions, however, washed with low salt concentrations and / or high temperatures.

In a further advantageous embodiment and further development of the invention, at least two different target nucleic acids, preferably 3 to 130 different nucleic acids, are read out at the same time. Advantageously, the carrier is then designed as a chip. In a further advantageous embodiment and development of the invention, the ligation is carried out at 37 ° C. In a further advantageous embodiment and development of the invention, the readout of the label is carried out by means of a branched DNA test and the reporter oligonucleotide is preferably a preamplifier. branched DNA), also known as the branched DNA assay, is a signal amplification assay for

Amplification of the reporter signal. For this purpose, a so-called "preamplifier" is used, which hybridizes directly or indirectly to the target nucleic acid to be detected. In subsequent steps, so-called "Amplifier" (in German: amplifier) hybridized to the "Preamplifier""preamplifier" and "Amplifier" are short single-stranded oligodeoxyribonucleotides The "Amplifier" are branched oligodeoxyribonucleotides bind Repor ^¬ termoleküle... Signal amplification is achieved by attaching multiple "amplifiers" to a preamplifier and each "amplifier" typically binds multiple reporter molecules.

By branching the "amplifiers", a high density of "amplifiers" on the "preamplifier" can be achieved, which leads to a high sensitivity of the "branched DNA" test. Thus, the sensitivity of the method is further increased by the use of the "branched DNA" test Depending on the marking used, in particular enzyme labeling or bDNA test, concentrations of the target molecules can be used, in particular when using an enzyme for test times of less than 30 minutes. For the use of a bDNA test, for test times of less than 45 minutes, concentrations of less than 100 fM are detectable

detectable.

The invention will be described below with reference to FIGS

Drawings still further explained.

Fig. 1 shows schematically the steps of the method for

Analysis of an RNA at times t1 to t5. Fig. 2 shows schematically a carrier with two capture oligonucleotides at time tl. 3 schematically shows a carrier with two catcher

Oligonucleotides, two reporter oligonucleotides and two miRNAs at time t2. schematically shows a support with two capture oligonucleotides and a reporter oligonucleotide at time t2 ^λ .

5 schematically shows a carrier with two catcher

Oligonucleotides and two reporter oligonucleotides, two miRNAs and the T4 DNA ligase at time t3.

6 shows schematically the miRNA with a capture

Oligonucleotide single strand and ligated to a reporter oligonucleotide single strand at time t4.

Fig. 7 shows schematically catcher oligonucleotide single strand after harsh wash conditions without miRNA presence at time t4 ^λ . FIG. 1 shows schematically the steps of the method for detection and quantification of a miRNA. In this example, the target nucleic acid is a miRNA named miR-16 5. MiR-16 5 is a miRNA that can reduce the expression of the anti-apoptotic protein Bcl-2 in lymphocytes. By way of example, the method can be subdivided into five steps in the ^{presence of} the miRNA 5 (steps t1, t2, t3, t4 and t5). In the absence of a target nucleic acid 5, the method in this example comprises the steps t1, t2 t4 ^λ and t5 ^λ .

In step t1, the provision of a support 1 with double-stranded capture oligonucleotides 2 immobilized thereon takes place (FIG. 2). Here, the first catcher Oligonucleotide partial strand 14 with a first end of a second single-stranded capture oligonucleotide overhang 3 via a spacer fixed to the support 1, that is immobilized. In particular, the spacer comprises six thymine bases. Alternatively, the first capture oligonucleotide partial strand 14 may be attached to the first end of the support with no oligonucleotide overhang present.

The first single-stranded capture oligonucleotide overhang 4 comprises an oligonucleotide sequence which is intended for targeting.

Nucleic acid miR-16 5 is partially complementary.

In the second exemplary method step at time t2 or t2 ^λ (FIGS. 3 and 4), the reporter oligonucleotide 6 is now added to the system and the biological sample is passed over the carrier 1.

In the case that the biological sample comprises the target nucleic acid miR-16 5, this step is shown in FIG. FIG. 3 shows schematically that the catcher

An oligonucleotide of the hybridized ^¬ Siert 2 with its first capture oligonucleotide overhang 4 having a first portion miR16. 5 Furthermore, it can be seen that a second portion of miR-16 hybridizes to a second reporter oligonucleotide overhang 8 which is complementary to the second portion of miR-16. The reporter oligonucleotide 6 comprises a label 7 on a second reporter oligonucleotide overhang 9. Alternatively, the two reporter oligonucleotide partial strands 16, 17 can have no overhang at the distal end viewed from the carrier, but can be approximately the same length.

In this example, the single-stranded portions of the capture oligonucleotide 4 and the reporter oligonucleotide 9 are complementary in that no free sequence portion remains free in the middle of the miRNA 5. Furthermore, the

Reporter oligonucleotide partial strand without labeling the complementary to a section of the target nucleic acid section. In the event that the biological sample does not comprise a target nucleic acid miR-16 5, the step at time t2 ^λ in Figure 4 is shown schematically. Even without the presence of a target nucleic acid 5, weak and un ^¬ specific bonds can arise between reporter oligonucleotide 6 and capture oligonucleotide 2.

In a next method step at time t3 (FIG. 5), ligase is now added to the system. In this example, it is a T4 DNA ligase 13. This ligase connects the first capture oligonucleotide partial strand 14 at the ligation site 10 with the miRNA 5 and the first repeat oligonucleotide partial strand 16. These compounds are covalent bonds. This means that the first capture oligonucleotide partial strand 14, the miRNA 5 and the first reporter oligonucleotide partial strand 16 are firmly connected to one another. For ligation of the individual strands 14, 5, 16, it is he ^¬ conducive that they directly adjoin one another. This is ensured by the fact that the two overhangs 4, 9 bind the miRNA 5 completely complementary.

In the event that no target nucleic acid present in the biological 5 ^¬'s test, the T4 DNA ligase can ligate the ends 13 is not of the capture oligonucleotide 2 and the reporter oligonucleotide 6 with the target nucleic acid. 5

The support 1 is then washed at a temperature of 55 ° C for 10 minutes with a salt-containing buffer solution. As a result, in the presence of the miR-16 5, the base pairings of the second capture oligonucleotide strand 15 and the second reporter oligonucleotide strand 17 are melted on ^¬ and the dissolved single-stranded oligonucleotides 15, 17 removed with the washing. This washing step is carried out with a low salt buffer.

If no miR-16 5 is present, unspecifically bound reporter oligonucleotide 6 will detach from the capture oligonucleotide 2 during washing of the support 1 at 55 ° C. The Re- Porter oligonucleotide 6 is thus removed from the carrier 1, so that no mark 7 remains on the carrier 1. Consequently, when reading the mark 7, no signal is measured. FIG. 7 shows the capture oligonucleotide 2 after this washing process.

Also in the event that a miRNA is present which is not complementary to the capture oligonucleotide 2 or reporter oligonucleotide 6, the reporter oligonucleotide 6 is washed away with the label 7 because there is no covalent bond after ligation. Thus, no signal is generated. This ensures a high specificity of the method for the respective target miRNA. Subsequently, the carrier 1 is brought to a readout temperature of 37 ° C at which the mark 7 on the carrier 1 from ^{¬ is} evaluated. As label 7, an enzyme is coupled to the reporter oligonucleotide 6. In this example, the enzyme is already bound to the reporter oligonucleotide as a label at the beginning of the detection procedure. Alternatively, the coupling of the enzyme in a further process step can be bound to the reporter oligonucleotide.

To read a signal, a substrate is placed on the carrier 1, which is reacted by the enzyme. The product of this re ^¬ action is electrochemically measured by a redox cycling of two electrodes. As the reporter enzyme, an esterase or an alkaline phosphatase can be used. As an alternative to a simple enzyme coupling to the reporter oligonucleotide 6, a signal amplification by means of a "branched DNA" tests can be done. In this case, the bDNA assays is in the ^¬ sem recordable the "preamplifier" subphase of the reporter, the to the target Nucleic acid is ligated.

Claims

claims

1. Device (20) for detecting at least one single-stranded target nucleic acid (5) in a sample, comprising:

at least one solid support (1) on which at least one double-stranded capture oligonucleotide (2) is immobilized, wherein the capture oligonucleotide (2) has a first capture oligonucleotide partial strand (14) and a second capture oligonucleotide (2) nucleotide substrand (15), wherein a first end of the first capture oligonucleotide substrand (14) is tightly attached to the support, and wherein the second capture oligonucleotide substrand (15) is a first capture oligonucleotide overhang (15). 4) on the side facing away from the carrier (1) side,

at least one double-stranded reporter oligonucleotide (6) having a first and a second reporter oligonucleotide substrand (16, 17), wherein a first end of the second reporter oligonucleotide substrand (17) has a first

single-stranded reporter oligonucleotide overhang (9),

and wherein the first capture oligonucleotide overhang (4) comprises at least a first oligonucleotide sequence which is complementary to a first target nucleic acid portion, and wherein the first reporter oligonucleotide overhang (9) comprises at least one second oligonucleotide Sequence which is complementary to a second target nucleic acid segment, to the first capture oligonucleotide partial strand (4) with the first target nucleic acid section and the first reporter oligonucleotide overhang (9) with the second Target nucleic acid section to hybridize;

- A read-out unit for reading a mark (7) of the first reporter oligonucleotide sub-strand (16) on the Trä ^¬ ger (1).

2. Device according to claim 1, wherein the first capture oligonucleotide overhang (4) and / or the first reporter oligonucleotide overhang (9) comprise eight to 25 bases.

3. Device according to one of claims 1 or 2, wherein the first end of the first capture oligonucleotide subunit (14) comprises six thymine bases.

4. Device according to one of claims 1 to 3, wherein the first capture oligonucleotide partial strand is formed as a chimera with at least one, in particular with four, RNA nucleotides at its 3 ^X -end.

5. A method for the detection and quantification of at least one single-stranded target nucleic acid (5) with the crotch ^¬ th:

a) providing at least one solid support (1) on which at least one double-stranded capture oligonucleotide (2) is mobilized in ^¬ , wherein the capture oligonucleotide (2) has a first capture oligonucleotide partial strand (14) and a second capture oligonucleotide Partial strand (15), wherein a first end of the first capture oligonucleotide partial strand (14) is tightly bound to the support and wherein the second capture oligonucleotide partial strand (15) has a first capture oligonucleotide overhang (4) has the side facing away from the carrier (1) side,

b) contacting the carrier (1) with at least one single-stranded target nucleic acid (5) and at least one double-stranded reporter oligonucleotide (6) in a first hybridization condition,

- wherein the reporter oligonucleotide (6) having first and ei ^¬ NEN second reporter oligonucleotide strand portion (16, 17) includes fully, wherein a first end of the second reporter

Oligonukleitd substrand (17) comprises a first single-stranded reporter oligonucleotide overhang (9),

and wherein the first capture oligonucleotide overhang (4) comprises at least a first oligonucleotide sequence which is complementary to a first target nucleic acid segment,

and wherein the first reporter oligonucleotide overhang (9) comprises at least one second oligonucleotide sequence which is complementary to a second target nucleic acid portion is such that the first capture oligonucleotide overhang (4) with the first target nucleic acid portion and the ers ^¬ th reporter oligonucleotide overhang (9) hybridizing to the second target nucleic acid portion;

c) ligating the target nucleic acid (5) to the first capture oligonucleotide partial strand (14) and to the first reporter oligonucleotide partial strand (16) by means of a ligase (13); d) first washing the support (1) under stringent washing conditions such that the support (1) is cleaned of unligated nucleic acids;

e) reading out a label (7) of the first reporter oligonucleotide partial strand (16) on the carrier (1).

6. The method of claim 5 with an RNA as the target nucleic acid.

7. The method of claim 6 with a miRNA (5) as RNA.

8. The method according to any one of claims 5 to 7 with a

T4 DNA ligase (13) as ligase.

9. The method according to any one of claims 5 to 8, wherein prior to ligating a second washing of the carrier (1) in mild

Washing conditions takes place.

10. The method according to any one of claims 5 to 9, wherein wenigs ^¬ least two different target nucleic acids, preferably three to 130 different nucleic acids are read from ^¬ simultaneously.

11. The method according to any one of claims 5 to 10, wherein the ligation is carried out at 37 ° C.

12. The method according to any one of claims 5 to 11, wherein the reading of the marker (7) by means of a "branched DNA" -

Tests carried out and the reporter oligonucleotide (6) vorzugswei ^¬ se a "preamplifier" is.