JP2010094075A - Method for detecting small-sized rna - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting a very fine amount of a small-sized RNA having a short strand length of ≤200 nucleotides, having a very resembling sequence and contained with a test specimen, a high sensitivity and a high accuracy. <P>SOLUTION: The method for detecting a small-sized RNA contained in the test specimen by using a selectively bonding substance-immobilized carrier having irregular parts on the carrier surface and immobilizing the selectively bonding substance on the nuchal planes of convex parts of the irregular part includes detecting a small-sized RNA by reacting a labeled body obtained by bonding a labeling material with at least one of the directional terminal of the small-sized RNA or a DNA having a complemental sequence to the small-sized RNA, with the selectively bonding material. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for detecting small RNAs such as microRNA and non-coding RNA molecules.

  Non-coding RNA (ncRNA) is a general term for RNAs that do not encode proteins, and is broadly classified into housekeeping RNAs and regulatory RNAs. Examples of the former housekeeping RNA include ribosomal RNA (rRNA), transport RNA (tRNA), small nuclear RNA (snRNA) involved in splicing, and small nuclear RNA (snoRNA) involved in rRNA modification. Are known. On the other hand, the latter regulatory RNA has attracted particular attention in recent years as a factor that plays an important role in elucidating biological functions, and regulates gene expression and intracellular distribution of RNA to suppress gene expression. Recently, it has been revealed that it plays an important role. The gene expression suppression mechanism in which this regulatory RNA functions is called RNA interference (RNAi), and was clarified in an experiment using nematodes in 1988, and then the existence of a similar mechanism in Drosophila and mammalian cells is also evident. It became. The ncRNA as a regulatory RNA has a chain length of about 20 to 25 bases, and its mechanism of action is the suppression of translation by microRNA (miRNA), the cleavage of target mRNA by snail interference RNA (siRNA), and the target DNA It is broadly divided into gene silencing through heterochromatinization of regions.

  miRNA is transcribed from endogenous miRNA as RNA (precursor) having a hairpin-like structure. This precursor is cleaved by a dsRNA cleaving enzyme (Drosha, Dicer) having a specific enzyme RNase III cleaving activity, then changed into a double-stranded form, and then becomes a single strand. One of the antisense strands is incorporated into a protein complex called RISC and is considered to be involved in the suppression of mRNA translation. Thus, since miRNA has different aspects at each stage after transcription, usually when miRNA is targeted (detection target), such as a hairpin structure, a double-stranded structure, a single-stranded structure, etc. Various forms need to be considered. miRNA consists of RNA of 15 to 25 bases, and its existence has been confirmed in various organisms.

  On the other hand, siRNA is cleaved from long-chain dsRNAs such as viruses, transposons, transgenes, and endogenous dsRNAs by a dsRNA cleaving enzyme (Dicer) having RNase III cleavage activity. Thereafter, one antisense RNA of dsRNA is incorporated into a protein complex called RISC or RITS, and is involved in mRNA degradation or transcriptional repression as a complex. Therefore, siRNA can have a double-stranded state as dsRNA and a single-stranded state forming a complex with RISC. Therefore, when siRNA is usually used as a target (detection target) It is necessary to consider various forms such as a long dsRNA, a cleaved siRNA, or a single strand.

  By the way, in general, in expression analysis using a nucleic acid-immobilized substrate (DNA chip), a test sample is placed on a DNA chip using a substrate on which probes using base sequences corresponding to hundreds to tens of thousands of genes are immobilized. By adding, the gene in the sample binds to the probe, and the amount of gene in the test sample can be known by measuring the amount of binding by some means. Selection of the gene corresponding to the probe to be immobilized on the DNA chip is free. However, when the detection target is the above-mentioned small RNA, the above-mentioned small RNA contained in a cell or tissue has a short chain length of 200 nucleotides or less and the sequence is very similar. It is difficult to distinguish small RNAs, and since the small RNAs contained in cells or tissues are very small, it is difficult to distinguish the small RNAs with high sensitivity.

  Accordingly, in the field of detecting and analyzing nucleic acids having a short chain length of the above-mentioned small RNA, establishment of an effective technique capable of detecting the above-mentioned small RNA with high sensitivity and high accuracy is an urgent issue.

  As one of the solutions, a method for measuring miRNA by adding a test sample labeled with the above small RNA to a DNA chip using a probe that specifically recognizes a guanine base contained in the above small RNA is proposed. (For example, refer to Patent Document 1). However, since the positions of guanine bases contained in the small RNA are usually diverse and the number of guanine bases contained in the small RNA is not constant, detection is possible depending on the number of guanine bases contained in the small RNA. The above-mentioned small RNA is produced that is difficult to

  In addition, a plurality of amino-modified adenine bases are enzymatically added to the 3 ′ end of the above small RNA, and a test sample in which a probe is labeled on the added amino-modified adenine base is added to a DNA chip, and miRNA is measured. A method has been proposed (see, for example, Patent Document 2). However, since the number of adenine bases imparted to the 3 ′ end varies and the number of probes labeled on the small RNA is not constant, the adenine base imparted to the 3 ′ end on the small RNA Depending on the number, miRNA or ncRNA that is difficult to detect is generated.

  In addition, a method of measuring miRNA by adding a test sample in which one cytosine base labeled with one probe at the 3 ′ end obtained by dephosphorylating the above small RNA is added to a DNA chip has been proposed ( For example, see Patent Document 3 and Non-Patent Document 1). However, since the size of one cytosine base is usually small and the size of the probe to be labeled is large, the above small RNA that is difficult to detect due to the steric hindrance of the labeled probe is generated only by adding one guanine base. . In addition, since the small RNA described above is in a very small amount, a sufficiently strong signal cannot be obtained with only one probe label, and the above small RNA is difficult to detect.

  In addition, a method is known in which small RNA is reverse-transcribed using a biotin-labeled random primer to detect biotin-labeled complementary DNA (see Non-Patent Document 2). Measurement may not be possible depending on the inspection data.

Further, a method is known in which the 3 ′ end of a small RNA is chemically modified and biotinylated for detection (see Non-Patent Document 3). However, measurement noise is introduced because of many modification work steps.
US Pat. No. 6,262,252 International Publication No. 2005/118806 Pamphlet US Patent Application Publication No. 2007/0172845 “Nucleic Acids Research”, 2004, Vol. 32, No. 11, e86. “Proc. Natl. Acad. Sci.”, June 29, 2004, vol. 101, no. 26, 9740-9744 “Nucleic Acids Research”, 2005, Vol. 33 (No. 2), e17

  The problem to be solved by the present invention is a method that can detect a very small amount of the above-mentioned small RNA with high sensitivity and high accuracy, the chain length of which is contained in a test sample is as short as 200 nucleotides or less and the sequence is very similar. Is to provide.

  The present invention has the following features.

  In the first embodiment, the present invention provides a test using a selective binding substance-immobilized carrier having a concavo-convex part on the surface of the carrier and a selective binding substance immobilized on the top surface of the convex part of the concavo-convex part. A method for detecting small RNA contained in a sample, comprising reacting a labeled substance having a labeled substance bound to at least one end of the small RNA or DNA having a sequence complementary to the small RNA with a selective binding substance A method for detecting small RNAs.

  In another embodiment, the method is for detecting small RNA, wherein the labeled body is a labeled body in which a labeling substance is bound to the 3 'end of small RNA or DNA having a sequence complementary to the small RNA.

  In another embodiment, small RNA is detected, wherein the labeled body is a labeled body in which a labeling substance is bound to the 3 ′ end of small RNA or DNA having a sequence complementary to small RNA via a linker sequence. It is a method to do.

  The present invention provides a method for detecting small RNA contained in a small amount of RNA sample with a high S / N ratio, and this makes it possible to detect small RNA with high sensitivity.

  The present invention will be described more specifically below.

  As used herein, small RNA refers to a range of less than 200 bases that constitutes many evolutionarily branched species including vertebrates, squirts, peninsulas, molluscs, annelids and arthropods. It means a certain RNA. Micro RNA (miRNA) is a single-stranded RNA of 15 to 25 bases contained in a small RNA, and its presence has been confirmed in various organisms.

  In the present specification, indications using abbreviations such as nucleotides and polynucleotides are in accordance with “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Japan Patent Office) and customary in the art. To do. Furthermore, “polynucleotide” is used as a nucleic acid including both RNA and DNA. The DNA includes any of cDNA, genomic DNA, and synthetic DNA. The RNA includes total RNA, mRNA, rRNA, tRNA, snRNA, snoRNA, miRNA, and synthetic RNA. In the present specification, the polynucleotide is used interchangeably with the nucleic acid.

  In the method for detecting small RNA of the present invention, a selective binding substance immobilized on a carrier surface is contacted with a solution containing small RNA that reacts with the selective binding substance or DNA having a sequence complementary to the small RNA. The small RNA is detected by observing the contact state.

  A complementary sequence (complementary strand, reverse strand) is a full-length sequence of a polynucleotide consisting of a base sequence constituting a small RNA, or a partial sequence thereof (referred to herein as a normal strand for convenience) A: T (U) A polynucleotide having a base-complementary relationship based on a base pair relationship such as G: C. However, such a complementary strand is not limited to the case where it forms a completely complementary sequence with the target positive strand base sequence, but has a complementary relationship that allows hybridization with the target normal strand under stringent conditions. There may be.

  In the present invention, as the selective binding substance-immobilized carrier, for example, those described in JP-A-2006-78197 can be used.

  The selective binding substance means a substance that can selectively bind directly or indirectly to a small RNA contained in a test sample. In the method for detecting small RNA of the present invention, specifically, nucleic acid derivatives such as DNA, RNA, PNA, LNA (Locked Nucleic Acid) can be used. Here, in the case of a nucleic acid, a derivative is a labeled derivative with a fluorophore, a modified nucleotide (e.g., a halogen or an alkyl such as methyl, an alkoxy such as methoxy, an alkoxy such as methoxy, a nucleotide or a base containing a group such as carboxymethyl, Chemically modified derivatives such as derivatives containing nucleotides etc. that have undergone double bond saturation, deamination, substitution of oxygen molecules with sulfur molecules, and the like.

  A single-stranded nucleic acid having a specific base sequence selectively hybridizes and binds to a single-stranded nucleic acid having a base sequence complementary to the base sequence or a part thereof. Applicable to substances. The selective binding substance used in the present invention may be a commercially available substance or may be obtained from a living cell or the like. Particularly preferred as the selective binding substance is a nucleic acid. Among these nucleic acids, nucleic acids called oligonucleic acids having a length of 10 to 100 bases can be easily artificially synthesized with a synthesizer. Furthermore, if it is less than 20 bases, 20-100 bases is more preferable from the viewpoint that the stability of hybridization is low.

  The small RNA to be detected may be miRNA (micro RNA). The information can be found by accessing, for example, https://www.sanger.ac.uk/Software/Rfam/mirna/index.shtml. In order to detect the target miRNA, a nucleic acid having a sequence complementary to the miRNA to be detected may be used as the selective binding substance.

  A DNA having a sequence complementary to a small RNA can be obtained by using a reverse transcriptase or the like from a small RNA contained in a test sample.

  Examples of test samples to be detected / diagnosed in this specification include natural sources such as tissue samples, whole organisms, cell cultures, body fluids, and the like.

  In the present invention, a labeled body labeled by binding a labeling substance to either at least the 3 'end or the 5' end of small RNA or DNA having a complementary sequence of small RNA is used. Preferably, a labeling substance is bound to the 3 'end. For binding of the labeling substance, an enzyme reaction, a chemical reaction, or the like can be used. Preferably, the reaction is performed using an enzyme. More preferably, a label is bound to the 3 'end of the small RNA.

  The labeling substance may contain a linker sequence that is a polymer of at least two or more nucleic acids. For the enzyme reaction, T4 RNA ligase, Terminal Deoxyditol Transfer, Poly A polymerase, or the like may be used.

  In the present invention, as the labeling substance that can be used, known substances used for labeling such as fluorescent dyes, phosphorescent dyes, and radioisotopes can be used. Preference is given to fluorescent dyes that are easy to measure and easy to detect signals. Specifically, cyanine (cyanine 2), aminomethylcoumarin, fluorescein, indocarbocyanine (cyanine 3), cyanine 3.5, tetramethylrhodamine, rhodamine red, Texas red, indocarbocyanine (cyanine 5), cyanine 5.5, known fluorescent dyes such as cyanine 7 and oyster.

  For detection of the fluorescent dye, a fluorescent microscope, a fluorescent scanner, or the like can be used.

  Moreover, you may use the semiconductor fine particle which has luminescent property as a label | marker. Examples of such semiconductor fine particles include cadmium selenium (CdSe), cadmium tellurium (CdTe), indium gallium phosphide (InGaP), silver indium zinc sulfide (AgInZnS), and the like.

  The method of binding a label to the end of a small RNA or a DNA having a sequence complementary to the small RNA can be performed using an enzymatic reaction. It is also preferable to use T4 RNA Ligase as the enzyme reaction.

  The invention is illustrated in more detail by the following examples. However, the present invention is not limited to the following examples.

Example 1
(DNA chip)
“3D-Gene (registered trademark) Human miRNA oligo chip” (Toray Industries, Inc .; TRT-XR311) was used as the selective binding substance-immobilized carrier.

(RNA labeling)
As a method for binding a labeling substance to the terminal, “miRCURY LNA microRNA Hy5 Power labeling kit” (Exicon; 208030-A) was used. As the test sample, FirstChoice Human River Total RNA (ABI; AM7960), which was confirmed to contain miRNA, was used. For each of the above totalRNA 100ng, 300ng, 500ng, and 1000ng, miRNA was labeled according to the manufacturer's recommended protocol.

(Hybridization)
All labeled miRNAs were each dissolved in “hybridization buffer” (Toray Industries, Inc .; TRT-XR301) and hybridized at 37 ° C. for 16 hours. After completion of hybridization, the DNA chip was subjected to a solution containing 0.5 × SSC and 0.1% SDS at 30 ° C., a solution containing 0.2 × SSC and 0.1% SDS at 42 ° C., and 0 ° C. at 30 ° C. Washed in continuous conditions with 0.05 × SSC solution.

(Measurement of gene expression level)
The DNA chip hybridized by the above method was scanned using a DNA chip scanner (ScanArrayLite, PerkinElmer Japan), an image was acquired, and the fluorescence intensity was digitized by GenePixPro5.0 (Molecular Device). Statistical processing is as follows: “Speed T.,“ Statistical analysis of gene expression microarray data ”, Chapman & Hall / CRC”, and “Courton H. C. et al.“ Abeginner's guide ”. I went there. That is, for the data obtained from the image analysis after hybridization, the logarithmic value was taken and the signal intensity was calculated for each miRNA.

  “3D-Gene Human miRNA oligo chip” (Toray Industries, Inc .; TRT-XR311) can detect about 800 types of microRNAs, from which RNA represented by SEQ ID NOs: 1, 2, 3, 4 Was selected and used for analysis.

  The results are shown in FIGS. 1, 2, 3, 4, and 5 as “Array 3”. The result of RNA of SEQ ID NO: 1 is shown in FIG. 1, the result of RNA of SEQ ID NO: 2 is shown in FIG. 2, the result of RNA of SEQ ID NO: 3 is shown in FIG. 3, and the result of RNA of SEQ ID NO: 4 is shown in FIG. FIG. 5 shows noise values of the example, the comparative example 1, and the comparative example 2. Comparative example 1 and comparative example 2 will be described later.

  Each figure is represented by a bar graph in which the signal intensity obtained from the results of Example 1 and Comparative Examples 1 and 2 is plotted on the vertical axis. From left to right, Array1-1000 ng (Comparative Example 1), Array2-3000 ng (Comparative Example 2), Array3-100 ng (when totalRNA is 100 ng in Example 1), Array3-300 ng (when totalRNA is 300 ng in Example 1), The results of Array3-500 ng (when totalRNA is 500 ng in Example 1) and Array3-1000 ng (showing results when totalRNA is 1000 ng in Example 1) are shown.

  1, 2, 3, and 4, it can be seen that in the examples, the signal intensity increases as the amount of total RNA increases. Further, it can be seen from FIG. 5 that the noise value is not related to the amount of total RNA.

Comparative Example 1
This was compared with the case of a DNA chip made of a flat glass substrate not provided with uneven portions.

  “Coated slide glass for DNA microarray” (Matsunami Glass) was used as the substrate. Under the following conditions, oligonucleotides were immobilized as selective binding substances (probe DNAs), respectively. DNA of complementary sequence (about 20 bases, 5 'terminal amination) was synthesized with the RNAs of SEQ ID NOs: 1, 2, 3, and 4. The DNA of SEQ ID NO: 1, 2, 3, 4 and the complementary sequence DNA are aminated at the 5 'end. This oligonucleotide was dissolved in Spotting Solution for DNA microarray (Matsunami Glass) so that the final concentration was 0.03 nmol and μL. A total of four spots were spotted on this solution using an arrayer (spotter) (manufactured by Nippon Laser Electronics; GeneStamp-II). The analysis chip obtained as described above was designated as “Array1”.

  For labeling of RNA, “miRCURY LNA microRNA Hy5 Power labeling kit” (Exicon Inc .; 208030-A) was used. As the test sample, FirstChoice Human River TotalRNA (ABI; AM7960), which was confirmed to contain miRNA, was used. The above total RNA of 1000 ng was used. Hybridization was applied to the surface on which the array 1 oligonucleotide was spotted using a micropipette. A gap cover glass (Matsunami Glass) was placed on the surface to which the test sample solution was applied, set in a hybridization chamber (Takara Bio Inc .; TX711), and reacted at 37 ° C. for 16 hours. The measurement of the gene expression level is the same as in the example. The result is shown as “Array 1-1000 ng” in FIGS.

  As can be seen from FIGS. 1, 2, 3, and 4, the signal intensity of Array 1-1000 ng is lower than that of 300 ng of total RNA in the Examples. Moreover, since the noise values are equivalent from FIG. 5, it can be said that Example 1 can be detected with a high S / N ratio even when using a smaller total RNA amount than Comparative Example 1.

Comparative Example 2
As a comparison with different labeling methods, a method of specifically recognizing and labeling a guanine base was compared. For labeling, Label ITmiRNA Labeling Kit, Cy5 (Takara Bio Inc .; V8610) was used, and for the above totalRNA of 3000 ng, miRNA was labeled according to the manufacturer's recommended protocol. Except for the labeling method, it is the same as in Example 1. The results are shown as “Array 2-3000 ng” in FIGS.

  As can be seen from FIGS. 1, 2, 3, and 4, the signal intensity of Array 2-3000 ng is lower in the Examples than when total RNA is 1000 ng (see FIGS. 1, 2, 3, and 4). Moreover, since the noise values are equivalent from FIG. 5, it can be said that Example 1 can be detected with a high S / N ratio even when using a smaller total RNA amount than Comparative Example 2.

  From the results of Example 1, Comparative Example 1 and Comparative Example 2 described above, miRNA was detected at a high S / N ratio from fewer test samples than Comparative Example 1 and Comparative Example 2 by using the method shown in Example 1. It can be said that it is possible.

The result of Example 1, the comparative example 1, and the comparative example 2 using RNA of sequence number 1 is represented. The result of Example 1, the comparative example 1, and the comparative example 2 using RNA of sequence number 2 is represented. The result of Example 1, the comparative example 1, and the comparative example 2 using RNA of sequence number 3 is represented. The result of Example 1, the comparative example 1, and the comparative example 2 using RNA of sequence number 4 is represented. The noise values of Example 1, Comparative Example 1, and Comparative Example 2 are represented.

Claims (3)

  A method for detecting small RNA contained in a test sample using a selective binding substance-immobilized carrier having a concavo-convex portion on a carrier surface and a selective binding substance immobilized on the top surface of the convex portion of the concavo-convex portion A small RNA comprising a selective binding substance and a label having a labeling substance bound to at least one end of the small RNA or a DNA having a sequence complementary to the small RNA. How to detect.   The method for detecting small RNA according to claim 1, wherein the labeled body is a labeled body in which a labeling substance is bound to the 3 'end of small RNA or DNA having a sequence complementary to the small RNA. The small RNA according to claim 1 or 2, wherein the labeled body is a labeled body in which a labeling substance is bound to the 3 'end of a small RNA or DNA having a sequence complementary to the small RNA via a linker sequence. How to detect.

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