JP2013099277A - Fluorescent probe for detecting rna with high accuracy - Google Patents

Abstract

PROBLEM TO BE SOLVED: To specifically detect an RNA having a similar sequence with high sensitivity.SOLUTION: Provided is a metastable double stranded probe capable of detecting a guanine-containing target nucleic acid, or a composition formable of the metastable double stranded probe, wherein the metastable double strand has at least one C/I base pair, and comprises a first strand having at least one cytosine residue and a second strand having at least one inosine residue (I) formable of a base pair with the cytosine residue, the second strand has a sequence corresponding to the target nucleic acid except the inosine residue, and the metastable double stranded probe is able to produce a stable double strand of the first strand with the target nucleic acid by performing strand exchange in the presence of the target nucleic acid.

Description

  The present invention relates to a metastable double-stranded probe or a composition capable of forming a metastable double-stranded probe, a target nucleic acid detection kit, and a target nucleic acid detection method.

  In recent years, much attention has been focused on RNA, particularly non-coding RNA that functions without being translated into protein in vivo. Among them, microRNA is a highly conserved short RNA molecule that acts as a key regulator in development, cell proliferation, differentiation, and cell cycle, and many miRNA sequences are currently known. There are still many unexplained parts regarding its functions. Also, as one of the next developments in research, analysis of microRNA expression levels between different tissues, developmental stage, and disease stage is closely watched. Therefore, it is necessary to develop a detection method for detecting these easily and accurately.

  Several products have been developed so far, but there are some problems. Methods for detecting RNA are generally classified into (i) polymerase chain reaction method (PCR method) and (ii) hybridization method. First, (i) is a method in which RNA is reverse-transcribed and then amplified and detected by PCR, but since it generally undergoes an amplification process, it is a method in which a great question remains in the correlation with the original RNA amount. In addition, both (i) and (ii) have the problem of recognizing RNA having a sequence very similar to the target RNA, so that highly selective detection of specific RNA has been achieved. There wasn't. In particular, microRNA is known to have a let-7 group, which has a similar sequence but is suggested to have a significantly different function (Non-Patent Document 1: Cell Research ( 2008) 18, 549-557.), It is important to be able to detect these expression levels with high accuracy. It has been shown that let-7a and let-7f could not be identified in TaqMan (registered trademark) MicroRNA Assays (applied biosystems) (Non-patent Document 2: PNAS, (2007) 104, 11400-11405, FIG. 1). ) In addition, Ambion's mirVana miRNA Probe has shown that Applied Biosystems' HP (TechNotes 12 (2)) was unable to discriminate single nucleotide differences in miRNA (http: //www.ambion) .com / jp / techlib / tn / 122 / 1.html, Fig. 2).

  Furthermore, conventional target RNA requires PCR, multiple times of hybridization and washing, labeling with a fluorescent dye, and the like, and skilled techniques are required to obtain stable results. In addition, some of the conventional products can identify the difference between 2 bases or 1 base, but this requires design for each target sequence, which requires time and cost.

  Patent Document 1 discloses a method for detecting a target RNA by hybridization using a competitive probe. However, in the case of RNA having a very similar sequence, sensitivity and specificity are not sufficient.

  Patent Document 2 discloses a method for detecting a double helix of a nucleic acid, but the detection sensitivity of RNA is not sufficient.

JP 08-116997 JP2011-173891

Cell Research (2008) 18, 549-557. PNAS, (2007) 104, 11400-11405

  The main object of the present invention is to provide a technique capable of highly sensitively and specifically detecting all target nucleic acids such as a group of RNAs having very similar sequences.

The present invention provides the following metastable double-stranded probe for high-precision detection of target nucleic acid (DNA or RNA) or a composition capable of forming a metastable double-stranded probe, for detecting target nucleic acid (DNA or RNA, particularly RNA) A kit and a method for detecting a target nucleic acid (DNA or RNA, particularly RNA) are provided.
Item 1: A metastable duplex probe capable of detecting a guanine-containing target nucleic acid or a composition capable of forming a metastable duplex probe, wherein the metastable duplex comprises at least one C / I base pair. The metastable duplex comprises a first strand having at least one cytosine residue and a second strand having at least one inosine residue (I) capable of base-pairing with the cytosine residue. The second strand has a sequence corresponding to the target nucleic acid except for the inosine residue, and the metastable double-stranded probe causes strand exchange in the presence of the target nucleic acid to stabilize the first strand and the target nucleic acid. A metastable double-stranded probe or a composition capable of forming a metastable double-stranded probe capable of producing a duplex.
Item 2: The composition capable of forming the metastable double-stranded probe or metastable double-stranded probe according to Item 1, wherein the first strand is DNA, RNA, LNA (BNA) or PNA.
Item 3: The composition capable of forming the metastable double-stranded probe or metastable double-stranded probe according to Item 1 or 2, wherein the first strand and / or the second strand are fluorescently labeled.
Item 4: The composition capable of forming the metastable double-stranded probe or metastable double-stranded probe according to any one of Items 1 to 3, wherein the target nucleic acid is RNA.
Item 5: The composition capable of forming the metastable double-stranded probe or the metastable double-stranded probe according to any one of Items 1 to 4, wherein the second strand includes one inosine residue.
Item 6: The metastable duplex probe or metastable duplex according to any one of Items 1 to 5, wherein the first strand has a fluorescent label and the second strand has a quencher label A composition capable of forming a probe.
Item 7: A target nucleic acid detection kit comprising the metastable double-stranded probe according to any one of Items 1 to 6 or a composition capable of forming a metastable double-stranded probe.
Item 8: The metastable double-stranded probe according to any one of Items 1 to 6 or a composition capable of forming a metastable double-stranded probe is brought into contact with a sample that may have a target nucleic acid, and the target nucleic acid and the first strand A method for detecting a target nucleic acid, comprising detecting the presence or absence of the formation of a stable double strand.

  According to the present invention, the target nucleic acid, in particular, RNA in general, including micro-RNA that has been attracting attention as having a function of regulating the expression of other genes in cells, has extremely high accuracy and high sensitivity. Can be detected. The present invention is superior to existing techniques in that (i) it does not include an amplification operation in RNA evaluation, and (ii) it can distinguish a difference in 1-base level.

  miRNAs are single-stranded RNAs of about 20-25 bases that regulate gene expression, and microRNAs are involved in many important biological processes from cancer to metabolism, development, and differentiation. It is said. Although miRNA has a group of RNAs with slightly different sequences, according to the present invention, even a single base difference in RNA, which has been difficult so far, can be accurately detected without amplification operation. It was. In particular, it was demonstrated by discrimination between Let-7a and let-7f that in fact, only the sequence of N = G in 20mer RNA (AUACACUGAANUGAAAACUG N = A, G, C, U) can be specifically detected. Furthermore, simultaneous detection in multiple colors can be easily achieved by changing the type of fluorescent dye and quencher to be introduced (for example, simultaneous detection in three colors or four colors is possible).

  Further, the presence or absence of target DNA / RNA can be detected simultaneously by the number of fluorescent dyes (wavelengths).

Non-Patent Document 2 shows that let-7a and let-7f could not be identified in TaqMan (registered trademark) MicroRNA Assays (applied biosystems). Ambion's mirVana miRNA Probe shows Applied Biosystems' HP (TechNotes 12 (2)), which describes that it was not possible to distinguish between single nucleotide differences in miRNA. Similar RNA sequences from has-let-7a to has-let-7i and mir-98 are shown. The structure of PNA is shown together with LNA (BNA) and RNA. Metastable double chain design. In FIG. 5, “Full match duplex” indicates a duplex in which the C / I base pair is replaced with a C / G base pair and becomes stable, and the full match duplex base pair is represented by C / G, A / It is composed of either T or A / U base pairs. Replacing any guanine base in the full match duplex with an inosine base can form a metastable duplex that is unstable for one hydrogen bond. It is a figure which shows how the equilibrium moves in the presence of a target nucleic acid and a non-target nucleic acid in the stable duplex (Full match), the metastable duplex, and the unstable duplex (Mismatch). When the target nucleic acid (Full match) is present, the equilibrium is inclined to form a stable double strand (Full match), and even if a non-target nucleic acid (Mismatch) is present, the metastable duplex is unstable (Mismatch). ) Is more stable, so substantially no unstable double strands (Mismatch) are produced. The case where a fluorescent label (F) is bound to the first strand and a quenching label (quencher, Q) is bound to the second strand is shown. Metastable duplexes cause strand exchange and emit fluorescence only when DNA and RNA with full match sequences are present. The difference in stability between DNA, RNA duplex and PNA / DNA, RNA duplex is shown. The difference in stability between Full match and Mismatch can be increased by using PNA (peptide Nucleic Acid). Metastable duplex probe design. Although FIG. 9 shows the case where the target nucleic acid is DNA, a metastable double-stranded probe can be similarly designed when the target nucleic acid is RNA. Evaluation of DNA discrimination ability using metastable double-stranded probes A method for designing the detection wavelength is shown. By changing the combination of the fluorescent substance and the quenching substance, two or more kinds of target nucleic acids can be detected in one test. A method for designing a detection base is shown. By changing the combination of the fluorescent substance and the quenching substance, two or more kinds of target nucleic acids can be detected in one test. Single base level target sequence recognition using two wavelengths. Recognition of RNA sequences ALDH2 full-length mRNA detection ALDH2 full-length mRNA detection target DNA detection region. It was demonstrated that 5 nM target DNA could be identified with 100 nM probe. It shows that let-7a and let-7f can be distinguished. The results of the gel shift assay (including controls) are shown.

  In this specification, examples of the target nucleic acid include DNA and RNA, and RNA is preferable. Examples of RNA include mRNA, tRNA, miRNA and the like, and miRNA is a particularly preferred target nucleic acid. RNA is mainly composed of 4 types of bases, and there are various types of RNA in cells ranging from more than a dozen bases to more than a few thousand bases. It is not easy to detect only. For example, let-7 family which is miRNA has similar sequences from has-let-7a to has-let-7i, mir-98 as shown in FIG. Although a probe capable of recognizing the sequence is required, it can be identified as shown in FIG. 18 by using the metastable double-stranded probe or the composition capable of forming the metastable double-stranded probe of the present invention.

  The length of the target nucleic acid may be 15 or more, preferably 20 or more, and the upper limit is not particularly limited, and a target nucleic acid having several hundred to several thousand bases can be identified.

  The metastable double-stranded probe of the present invention may be any one in which two strands are linked by a base pair of nucleobase such as adenine, guanine, cytosine, thymine, uracil, inosine, and the like, DNA, RNA, LNA (BNA) Any of PNA and the like may be used. LNA (BNA), PNA, etc. are preferably applied to the first strand containing cytosine residues, and the second strand is the same type of nucleic acid (DNA or RNA) as the target nucleic acid except that G is changed to I Preferably there is. The structures of RNA, LNA (BNA) and PNA are shown in FIG. LNA (BNA), PNA, etc. have stability between a stable duplex (full match duplex) hybridized with a target nucleic acid and an unstable duplex (mismatch duplex) bound with a non-target nucleic acid of similar sequence. The difference increases and sequence discrimination ability is improved (FIGS. 5-8).

  The probe of the present invention may be used by forming a metastable double-stranded probe in advance, or may be used as a composition comprising a first strand and a second strand constituting the metastable double-stranded probe. The composition forms a metastable double-stranded probe when reacted alone in a solution, but when reacted with a target nucleic acid, the first strand and the second strand are formed if a double strand is formed in advance. Since the target nucleic acid binds to the first strand after the strand separation of, for example, the first strand and the second strand are synthesized and mixed in powder form, or separate solutions are prepared in the sample. The conditions may be such that a metastable double-stranded probe can be formed by mixing together. In the composition of the present invention, the first chain containing cytosine and the second chain containing inosine may be used in equimolar to equivalent amounts, or the second chain may be used in excess. When the first strand is fluorescently labeled and the second strand is quenched, the second strand is preferably used in an equimolar to excess amount.

  In the present specification, the base pair of cytosine residue (C) and inosine residue (I) is referred to as “C / I”. Similarly, the base pair of adenine residue (A) and uracil residue (U) is “A / U”; the base pair of adenine residue (A) and thymine residue (T) is “A / T”; The base pair of the residue (C) and the guanine residue (G) is expressed as “C / G”, respectively.

  “Metastable duplex” refers to a duplex having at least one cytosine residue (C) and inosine residue (I) base pair (C / I). A metastable duplex usually has one or two, especially one C / I, but as the metastable duplex becomes longer, it can have more than one C / I. When a metastable duplex has 30 or fewer base pairs, it usually has one C / I base pair. When the length of the metastable duplex is 50 bases or longer, such as 100 bases or longer, the number of inosine residues can be increased in proportion to the length. Further, when the G / C content in the metastable duplex is high, the number of inosine residues can be increased accordingly.

  “Full match duplex” refers to a duplex composed of either C / G and A / T or a combination of two base pairs, C / G and A / U. For example, when the target nucleic acid is RNA, a combination of two base pairs C / G and A / U, and when the target nucleic acid is DNA, a combination of two base pairs C / G and A / T A stable duplex may be constructed.

  “Mismatch duplex” means that one or more base pairs contained in the first strand sequence do not form one or more complementary strands. The position of the mismatch included in the “mismatch duplex” may be the end or the center.

  “The second strand has a sequence corresponding to the target nucleic acid excluding the inosine residue” means that the second strand nucleic acid residues (A, T, C, G, U) other than the inosine residue are the target. It means a base that forms a nucleic acid A / T or A / U, C / G base pair.

  The position of inosine residue (I) in the second strand may be at the end or in the middle, but having an inosine residue at or near the center improves sensitivity and specificity. This is preferable.

  Of the guanine-containing target nucleic acids, those in which the guanine site is replaced with an inosine residue is the second strand. When the target nucleic acid is DNA, the second strand is DNA, and when the target nucleic acid is RNA, the second strand Is preferably composed of RNA.

  As shown in FIG. 5, when the target nucleic acid coexists with the metastable double-stranded probe of the present invention, the duplex of the target nucleic acid and the first strand containing a cytosine residue becomes more stable. Strand exchange between the second strand of the duplex and the target nucleic acid occurs. The duplex between the target nucleic acid and the first strand containing a cytosine residue is a metastable duplex in which C / I is replaced by C / G. Become. On the other hand, when non-target nucleic acids having different sequences coexist in any position of the metastable double-stranded probe of the present invention, the duplex of the non-target nucleic acid and the first strand containing a cytosine residue is metastable duplex. Since it is a less stable duplex (Mismatch duplex), no strand exchange occurs, and the metastable duplex exists as it is.

  The strand exchange between the sample that may contain the target nucleic acid and the metastable double-stranded probe of the present invention may be performed at room temperature, but is preferably performed under heating conditions. The heating conditions are, for example, 50 to 100 ° C, preferably about 60 to 95 ° C, more preferably about 70 to 90 ° C. The chain exchange reaction time is about 0.1 minute to 1 hour, preferably about 0.2 to 30 minutes, more preferably about 0.3 to 10 minutes, still more preferably about 0.4 to 5 minutes, especially 0. About 5 to 3 minutes. Since the chain exchange reaction takes longer as the temperature is lower, the reaction conditions can be appropriately selected.

  In this way, since only the target nucleic acid causes strand exchange with the metastable double-stranded probe, the difference in one base can be detected sensitively, and the target nucleic acid can be detected with high sensitivity and specificity.

  The first strand and the second strand constituting the metastable double-stranded probe of the present invention may be composed only of a base sequence, but in order to improve water solubility, a water-soluble group is provided at one or both ends. Can be combined. Examples of such water-soluble groups include amino acids or peptides, polyether groups such as polyethylene glycol (PEG), sugar chains, and the like. When an amino acid or peptide is bound to a nucleic acid, a basic amino acid (Arg, Lys, His, preferably Lys) or a peptide containing a basic amino acid (for example, KKK) can neutralize charge and impart water solubility. Although it is also preferable, acidic amino acids (Glu, Asp, etc.) can also be used to impart water solubility. Furthermore, neutral and water-soluble amino acids such as Ser can be used, and acidic, neutral and basic amino acids can be used in appropriate combination. Moreover, in order to adjust the positional relationship between the fluorescent substance and the quencher, for example, an arbitrary spacer such as AEEA may be combined.

As shown in FIG. 6, an appropriate fluorescent dye is modified on the first strand complementary to the sequence of the target nucleic acid (may be PNA, DNA, RNA, LNA (BNA), etc.). The quencher is modified to a second strand that is more unstable than the target RNA (specifically, a decoy RNA whose stability is reduced by one hydrogen bond by replacing G with I). Prepare things. By doing so, when the target RNA is not present, the first strand and the second strand (decoy RNA) form a complementary strand, and thus the fluorescence is turned off. If the target RNA is present there, strand exchange occurs and fluorescence is restored. It should be noted that when there is RNA (pseudo RNA) that differs by only one base compared to the target RNA, decoy RNA is more stable and no strand exchange occurs. In other words, compared to the conventional method, even if pseudo RNA other than the target RNA coexists, it does not respond to them, and therefore can be detected with high selectivity. Features of preferred embodiments of the invention labeled with such fluorescent and quenchers for metastable duplex probes include the following:
(i) The presence of the target RNA can be evaluated by a simple operation only by measuring the fluorescence intensity.
(ii) Use an inosine-guanine base pair that is unstable by only one hydrogen bond than the target RNA as a metastable duplex probe.
(iii) RNA can be specifically detected even in the presence of single-stranded or double-stranded DNA containing the target sequence.
(iv) No treatment for target RNA (labeling with PCR amplified fluorescent dye) is required.
(v) The design of the probe for the target RNA is easy.

  FIG. 9 shows a method for designing a metastable double-stranded probe or a second-strand probe of the composition. As shown in FIG. 9, the second strand can be easily designed by changing at least one G of the target nucleic acid to I.

  The metastable double-stranded probe preferably has a fluorescent label. FIG. 7 shows the case where the metastable duplex probe has a fluorescent label. Fluorescent substances for labeling include FAM (5- or 6-carboxyfluorescein), VIC, NED, fluorescein, FITC, IRD-700 / 800, CY2, CY3, CY5, CY3.5, CY5.5, HEX, TET, TAMRA, SNARF, SNAFL, Naphtofluorescein, JOE, ROX, BODIPY TMR, Oregon Green, Rhodamine Green, Rhodamine Red, Texas Red, Yakima Yellow, Alexa Fluor PET (Alexa Fluor) PET, BioSearch Blue (trademark), Marina Blue (Registered trademark), Bothel Blue (registered trademark), Alexia Fluore (registered trademark), 350 FAM (registered trademark), SYBR (registered trademark) Green 1, Fluorescein, Evergreen (registered trademark), Alexia Fluore (registered trademark) 488 JOE ( Trademark), V C (TM), HEX (TM), TET (TM), CAL Fluor (R) Gold 540, Yakima Yellow (R), ROX (TM), Calfluore (R) Red 610, Cy3.5 (Trademark), Texas Red (trademark), Alexia Fluore (trademark), 568Cy5 (trademark), Quasar (trademark) 670, Light Cycler red 640 (trademark), Alexia fluore 633 Quasar (trademark) 705, Light Cycler Red 705 (trademark), Alexia Fluore (trademark) 680, SYTO (trademark) 9, LC Green (trademark), LC Green (trademark) plus, Evergreen (trademark) ), Naphthalene, pyrene, perylene, anthracene, pentacene, dansyl Dansyl), coronene, and the like quantum dots.

The metastable double-stranded probe is preferably a combination of a fluorescent substance and a quenching substance (quencher) that is quenched when the duplex is bound, and emits light when strand exchange with the target nucleic acid occurs (FIG. 5). ). Such combinations include Cy5 and BHQ3, Cy3 and BHQ2, FAM and BHQ1, Cy2 and BHQ1, Coumarin and Dabsyl, BHQ1 and TET, BHQ2 and HEX, BHQ2 and JOE, BHQ0 and Alexa (registered trademark) 350, BHQ0 and Pacific Blue, BHQ0 and Dansyl, BHQ0 and CY2, BHQ1 and FAM, BHQ1 and JOE, BHQ1 and Orenge Green (registered trademark), BHQ2 and TAMRA, BHQ2 and CAL Fluore Red, BHQ2 and ROX, BHQ2 and Quasar2 570B And Quasar670, BHQ3 and CAL Fluore Red, BHQ3 and Quasar670
Etc.

  As shown in FIG. 11, the detection wavelength can be arbitrarily selected by selecting the combination of the fluorescent substance and the fluorescence wavelength.

  As shown in FIG. 12, the detection base can be easily changed by using a plurality of combinations of fluorescent substances and quenching substances and applying different detection bases to each.

  The fluorescent material may be bound to the first strand and the quencher may be bound to the second strand, but the fluorescent material may be bound to the second strand and the quencher may be bound to the first strand. According to such a configuration, the presence or absence of the target nucleic acid can be detected by measuring fluorescence.

Conventionally, for example, the following products (i) to (v) are commercially available as RNA detection kits. When the metastable double-stranded probe or composition of the present invention is further added to these kits, it can be reacted under the same conditions as in conventional kits, and sensitivity and specificity can be further improved.
(i) Applied Biosystems: TaqMan® MicroRNA Assays
(ii) Ambion: mirVana miRNA Probe
(iii) EXIQON: miRCURY probe (miRCURY LNA microRNA Array)
(iv) Veritas: QuantiGene 2.0 miRNA Assay
(v) TORAY: miRNA Oligo chip

  Metastable double-stranded probes or compositions can measure target nucleic acids in the range of at least 1 nM to 1 μM, but can also detect target nucleic acids at lower concentrations by improving the sensitivity of fluorescent substances. Is possible.

  The probe of the present invention is preferably present in the detection system in an amount equivalent to 100 times (preferably 3 to 10 times) or more of the target nucleic acid, as shown in FIG. When the concentration is 100 nM, the target nucleic acid can be detected as a significant difference in the concentration range of 5 nM-1 mM.

  These indicate that even when the target nucleic acid such as RNA to be detected is different by only one base, it can be detected.

  Add the inosine residue-containing second strand (DNA or RNA) or a metastable double-stranded probe containing it to the sample containing the target nucleic acid (DNA or RNA), and anneal the conventional microarray to improve the accuracy of the microarray. Improvement can be expected. In addition, the accuracy of various kits using hybridization such as Southern blotting and Northern blotting can be expected.

  The metastable double-stranded probe of the present invention eliminates the need for a washing operation by binding an appropriate fluorescent label, can detect a target nucleic acid in a living cell, and can detect a target nucleic acid in a cell disruption solution You can also.

  The detection method of the present invention is very sensitive and specific, and can detect a target nucleic acid in one cell.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, it cannot be overemphasized that this invention is not limited to these Examples.

Example 1
(1) PNA synthesis The peptide was synthesized by a peptide solid-phase synthesis method (Fmoc method) using an automatic peptide synthesizer (SHIMADZU PSSM-8). FmocLys (Boc) -Alko-PEG-Resin (0.21 mmol / g, 100-200 mesh, 1% DVB) 10 mg was used as a carrier, and each PNA monomer and amino acid were elongated. Respective monomers Fmoc-A (Bhoc) aeg-OH (10.0 mg, 5 eq.), Fmoc-G (Bhoc) aeg-OH (10.2 mg, 5 eq.) Dissolved in NMP (N-methylpyrrolidone), Fmoc-C (Bhoc) aeg-OH (9.6 mg, 5 eq.), Fmoc-Taeg-OH (7.0 mg, 5 eq.), Fmoc-Lys (Boc) -OH (4.9 mg, 5 eq.), Fmoc -AEEA-OH (4.9 mg, 5 eq.) Was reacted in 0.3 M HBTU / HOBt (35 ul, 5 eq.), 1M N, N-diisopropylethylamine (23 ul, 5 eq.) For 30 minutes. Deprotection of the Fmoc protecting group was performed twice using 30% piperidine (500 ul). After the synthesis, the product was cut out with 1 ml of 90% trifluoroacetic acid (TFA) and 10% m-cresol for 3 hours, deprotected, precipitated with cold ether, and purified by HPLC.

  A list of synthetic PNAs and MS results are shown in Table 1.

(2) DNA synthesis Gene Design Co., Ltd. was requested to synthesize DNA with the following sequences.

  A list of synthetic DNAs and purification methods are shown in Table 2 below. Confirmation of DNA was performed by MS (mass spectrometry).

RNA synthesis ALDH2 cRNA (rI) -BHQ3 shown in Table 3 was commissioned to Gene Design Co., Ltd.

  Let-7a dI-RNA-BHQ3 in Table 3 was synthesized using an automatic DNA / RNA synthesizer (Applied Biosystems 392 DNA / RNA synthesizer). After the synthesis, it was cut out in 28% ammonia water, deprotected (room temperature, 12 hours), and lyophilized. This was treated with 1M tetrabutylammonium fluoride (in THF, 50 ul) at 60 ° C. for 1 hour in anhydrous DMSO (100 ul) and purified by HPLC.

  DNA template (50 uM, 2 ul) with T7 promoter sequence (TAATA CGACT CACTA TAGGG) added 5 'to the target RNA sequence, 100 mM DTT (10 ul), 10 x reaction buffer (10 ul), 25 mM rNTP (30 ul), T7 RNA polymerase (10 ul) and purified water (38 ul) were reacted at 37 ° C. for 2.5 hours. After the reaction, purification was performed with 8% PAGE.

RNA detection
Mix 10 uM PNA (1 uL), 30 uM decoy RNA (1 uL), 10x PBS buffer (1 uL) 1 mg / mL BSA (1 uL) MQ water (6 uL), and heat at 80 ° C for 1 minute It was. Then, the 10x probe solution was adjusted by cooling at room temperature. 2 uL of this 10 × probe solution was added to 18 uL of each target RNA sample and heat-treated at 80 ° C. for 1 minute. Then, it cooled at room temperature and measured the fluorescence intensity of each sample by TECAN infinite 200 PRO. The results are shown in FIG. Further, it was confirmed that the detection of ALDH2 full-length mRNA shown in FIG. 15 was also possible (FIG. 16).

DNA detection
10 uM PNA (1 uL), 30 uM decoy DNA (1 uL), 10x PBS buffer (1 uL) 1 mg / mL BSA (1 uL) water (6 uL) were mixed and heat-treated at 95 ° C for 3 minutes . Then, the 10x probe solution was adjusted by cooling at room temperature. 2 uL of this 10 × probe solution was added to 18 uL of each target DNA sample and heat-treated at 95 ° C. for 3 minutes. Then, it cooled at room temperature and measured the fluorescence intensity of each sample by TECAN infinite 200 PRO. The evaluation results of DNA discrimination ability are shown in FIGS. 10, 11, 12, and 13, respectively.

  As a result of the DNA detection probe, the metastable double-stranded probe of the present invention was successfully used to identify a difference of only one base out of 20 bases of the DNA base sequence (FIGS. 10, 11, 12, and 13). ).

The metastable double-stranded probe of the present invention or the composition capable of forming the probe has the following advantages.
-The excitation wavelength / detection wavelength of the probe can be selected arbitrarily.
-Target sequence and detection base can be designed easily.
-A large number of target nucleic acids can be detected simultaneously.

  A sequence-selective probe of RNA can be easily obtained simply by changing a metastable double-stranded probe containing DNA (eg, PNA / DNA) to a metastable double-stranded probe containing RNA (eg, PNA / RNA).

Gel shift assay (DNA)
Mix 10 uM DNA (2 uL), PNK (10 U / uL, 0.8 uL), 10x PNK buffer (2 uL), γ ³² P-ATP (1.66 uM, 2 uL) water (13.2 uL) at 37 ℃ Incubated for 2 hours. 30 uL of purified water was added thereto, 100 uL of phenol / chloroform treatment was performed, and the aqueous phase was purified with Bio-spin 6 columns substituted with 1 × PBS. This was added to unlabeled DNA 111 nM at 1000 count / uL to obtain a target DNA solution.

  10 uM PNA (1 uL), 30 uM decoy DNA (1uL), 10x PBS buffer (1uL) 1 mg / mL BSA (1 uL) 30% sucrose (w / v) (2 uL) MQ water (4 uL ) And heat-treated at 95 ° C. for 3 minutes. Then, the 10x probe solution was adjusted by cooling at room temperature.

  2 uL of this 10 × probe solution was added to 18 uL of each target DNA solution (6% sucrose), and heat treatment was performed at 95 ° C. for 3 minutes. Then, it was left at room temperature for 30 minutes. 4 uL of this solution was electrophoresed on a 15% polyacrylamide gel (1x TBE buffer) at 150 V for 45 minutes, and then a SCAN image was obtained with a phosphor imager STORM820 manufactured by GE healthcare.

  The image result of the acrylamide gel by the gel shift assay is shown in FIG.

The composition of 1x PBS buffer is shown below.
KCl 200 mg / L
KH ₂ PO ₄ 200 mg / L
NaCl 8 g / L
Na ₂ HPO ₄ 1.15 g / L

  Preferred probes of the present invention can be designed by the same method for both DNA and RNA, there is almost no restriction on the target sequence, the design is simple, the sequence selectivity is high, the detection wavelength is not restricted, the fluorescent dye (wavelength ), The presence or absence of target DNA / RNA can be detected at the same time, and it is not necessary to examine the line condition according to the target nucleic acid, and it is suitable for DNA or RNA detection kits and contracted services.

Claims (8)

A metastable duplex probe capable of detecting a guanine-containing target nucleic acid or a composition capable of forming a metastable duplex probe, wherein the metastable duplex has at least one C / I base pair; The metastable duplex is composed of a first strand having at least one cytosine residue and a second strand having at least one inosine residue (I) capable of base-pairing with the cytosine residue. The second strand has a sequence corresponding to the target nucleic acid except for the inosine residue, and the metastable double-stranded probe causes strand exchange in the presence of the target nucleic acid, thereby stabilizing the first duplex and the target nucleic acid duplex. A metastable double-stranded probe or a composition capable of forming a metastable double-stranded probe. The composition capable of forming a metastable double-stranded probe or metastable double-stranded probe according to claim 1, wherein the first strand is DNA, RNA, LNA (BNA) or PNA. The composition capable of forming a metastable double-stranded probe or metastable double-stranded probe according to claim 1 or 2, wherein the first strand and / or the second strand are fluorescently labeled. The composition which can form the metastable double stranded probe or metastable double stranded probe in any one of Claims 1-3 whose target nucleic acid is RNA. The composition capable of forming a metastable double-stranded probe or a metastable double-stranded probe according to any one of claims 1 to 4, wherein the second strand contains one inosine residue. The metastable double-stranded probe or metastable double-stranded probe according to any one of claims 1 to 5, wherein the first strand has a fluorescent label and the second strand has a quencher label. A formable composition. A kit for detecting a target nucleic acid, comprising a metastable double-stranded probe or a composition capable of forming a metastable double-stranded probe according to any one of claims 1 to 6. A metastable double-stranded probe according to any one of claims 1 to 6 or a composition capable of forming a metastable double-stranded probe is brought into contact with a sample that may have a target nucleic acid, thereby stabilizing the target nucleic acid and the first strand. A method for detecting a target nucleic acid, comprising detecting the presence or absence of formation of a duplex.