JP4868391B2 - Intelligent nucleic acid sequence sensing system using transcription mechanism - Google Patents

Description

  The present invention relates to a method for producing a nucleic acid probe having sequence information of a sample nucleic acid and a method for detecting a target nucleic acid in a sample. The present invention also relates to a nucleic acid probe and kit for use in the above method.

  Nucleic acid molecules, which are biopolymers, not only serve as a storage medium for genetic information, but also have the ability to recognize nucleic acids, proteins, small organic compounds, or small organisms by forming three-dimensional structures that become physiological activities and binding pockets as enzymes. Etc. In recent years, research on drug discovery and medical applications of nucleic acid molecules programmed to perform various functions according to the purpose using the information processing function of nucleic acid molecules Has attracted attention as an application field.

  The functions of these nucleic acid molecules can be broadly divided into “programming functions”, “memory functions”, “functions as physiologically active substances”, “functions as other binding functional substances”, etc. Significant progress has been made in the use of "diagnostic technology".

  Among the “programming (information processing) functions”, Benenson et al. Used a restriction enzyme that recognizes special sequences as hardware to program information into nucleic acid molecule sequences and apply them to diagnosis and treatment. Developed a system that uses nucleic acid molecules that release DNA drugs by determining the presence of RNA that is specifically expressed in prostate cancer and small cell lung cancer. However, the function has not been confirmed in vivo (Non-patent Document 1).

  Among the “memory functions”, as a method of learning specific sequence information in the sequence of a nucleic acid molecule and using it as a memory, Chen et al. Utilize random interactions between DNAs that form inaccurate hybridization. We have succeeded in developing a DNA memory capable of learning and reading out, and proposed that it can be used for analysis and classification of expressed genes (Non-patent Document 2). However, a method for producing a memory capable of storing RNA has not been specifically presented. If RNA can be collected from saliva and a similar memory can be created, disease areas where RNA diagnosis is effective will expand in the future, leading to early detection and early treatment by simple diagnosis with low invasiveness. Expected (Non-Patent Document 6).

  As "function as a physiologically active substance", nucleic acid has not only a diagnosis but also an effect as a therapeutic agent. Among nucleic acid molecules having a specific sequence, those that have been confirmed to be therapeutically effective as DNA drugs are already in practical use. Vitravene (antisense drug; an ssDNA drug that inhibits the synthesis of oncogene protein MDM2, which plays an important role in the inactivation of p53 tumor suppressor protein, and is effective in treating retinitis by CMV (cytomegalovirus) in AIDS; Non-patent document 3), Obrimersen (antisense medicine; non-patent document 4) for advanced malignant melanoma. Further, among nucleic acid molecules having a specific sequence, an RNA aptamer (NX1838) against vascular endothelial growth factor (VEGF), which is a therapeutic agent for senile macular degeneration, has been confirmed to be therapeutically effective as an RNA drug. (Non-Patent Document 5). RNA drugs, like DNA, can be improved in function and mass-produced, and have the advantage that immune exclusion does not occur compared to antibody drugs. However, RNA drugs have the problem of being inferior to DNA in terms of stability. In recent years, it has attracted particular attention because it is decomposed in a certain time and is easy to control in time.

In addition, among the nucleic acid molecules as "other binding functional substances", there is a malachiteline aptamer created by Grate et al.
Non-patent document 7). Malachite green aptamer is a kind of aptamer, also called binding functional nucleic acid, has the ability to form a three-dimensional structure as a binding pocket and bind to malachite green (MG), and is released when malachite green is excited with a laser. Developed for the purpose of RNA-CALI (chromophore-assisted laser inactivation), which inactivates RNA incorporating a malachite green aptamer sequence using active oxygen and can analyze the function of target genes at the transcriptional level. (Non-Patent Document 7)
). Other properties of malachite green aptamers are known to enhance the fluorescence of malachite green when malachite green is incorporated into the binding pocket. By incorporating a module sequence that can recognize adenosine triphosphate (ATP), which plays an important role, and theophylline, a bronchodilator, it can be used as a sensor that can detect these substances by fluorescence. Is shown (Non-patent Document 8). In addition, a T7 sense DNA containing a part of the T7 promoter sequence and the aptamer template as a complementary strand as a minimum DNA sequence necessary for transcription of the malachite green aptamer, and a sequence and aptamer complementary to the T7 promoter sequence The sequence of the antisense DNA containing the template sequence has already been clarified (Non-patent Document 9).

  As a method for detecting RNA by fluorescence enhancement, in addition to a method using malachite green, 2′-O-methyl RNA (OMUpy) in which pyrene known as an organic electroluminescence device is introduced into the 2 ′ hydroxyl group of the sugar ring of uridine is used. ) Has also been synthesized, and a method for detecting RNA having a sequence complementary to 2′-O-methyl RNA using this has already been clarified (Non-patent Document 10).

  In addition to this method, for example, RNA can be detected by measuring radiation by incorporating a substrate labeled with a radioactive substance by a method known to those skilled in the art.

  In addition, as a method for detecting that RNA was transcribed regardless of fluorescence, biotinylated aptamer having a function of binding to a target protein by introducing a biotinylated substrate (such as yTP) was avidinized. An aptamer is efficiently immobilized using an avidin-biotin bond on a sensor chip of an interaction measuring device (for example, BIACORE) using a crystal oscillator or surface plasmon resonance, and the binding activity with a target protein is achieved using the immobilized aptamer. A method for detecting the presence of a target RNA by observing is known (Non-patent Document 11).

Furthermore, it has been reported that breast cancer and oral cancer can be judged non-invasively, simply and accurately based on changes in the amount of saliva mRNA transcribed, and research into diseases that can be diagnosed with mRNA in saliva will progress in the future. It is considered to be (Non-Patent Document 6). In Non-Patent Document 6, the target that can be diagnosed with mRNA in saliva is not limited to breast cancer and oral cancer, and there is a possibility that the mRNA pattern in saliva provides information on other cancers and frequent diseases. Has been suggested. Saliva reflects the state of the blood, and it is expected that many diseases can be found by saliva testing. Therapeutic and diagnostic research targeting the pathogenic genes of intractable diseases is also progressing. Based on recent results of brain science, genes that are specifically expressed in the brain in psychiatric and neurological diseases such as schizophrenia and Alzheimer's disease Analysis is progressing. In order to prepare an early diagnosis and early treatment system based on RNA patterns in saliva, comprehensive research is required to search for diseases that can diagnose pathogenic gene changes from RNA patterns in saliva. Saliva can be collected easily and in large quantities, and it has been confirmed that specificity and sensitivity are better for breast cancer and oral cancer than for blood diagnosis (Non-Patent Document 6). There are about 3000 types of mRNA in saliva, of which about 280 are unique to the saliva of healthy individuals. To determine cancer and systemic diseases from RNA patterns in blood, samples of mRNA patterns specific to cancer and systemic diseases are collected from patient populations out of the remaining 2700 types of mRNA and determined by large-scale analysis. There is a need. Currently, data obtained using a DNA chip is analyzed using a regression tree model or a logistic regression model, but it is difficult to determine an mRNA pattern as a biomarker that can be used for disease determination. This is because not only various factors are involved in mRNA fluctuations, but it is also possible that multiple diseases may develop. In order to determine morbidity and morbidity risk and realize tailor-made medical care, it is desired to realize an inexpensive, simple, and sensitive analysis system that can perform comprehensive analysis.

Benenson, Y .; Gil, B .; Ben-Dor, U .; Adar R .; Shapiro, E .; Nature 2004, 429, 423-429. Chen, J .; Deaton, R .; Wang, Y-Z .; LNCS 2004, 2943, 145-156. Holmlund, J. T .; Ann. NY Acad. Sci. 2003, 1002, 244-251. Klasa, R. J .; Gillum, A. M .; Klem, R. E .; Frankel, S. R .; Nucleic Acid Drug Dev. 2002, 12, 193-213. Drolet, DW .; Nelson, J .; Tucker, CE .; Zack, PM .; Nixon, K .; Bolin. R .; Judkins, MB .; Farmer, JA .; Wolf, JL .; Gill, SC .; Bendele, RA .; Pharm Res. 2000, 17 (12), 1503-1510. Li Y .; St. John, MAR .; Zhou, Z .; Kim, Y .; Sinha, U .; Jordan, RCK .; Eisele, D .; Abemayor, E .; 2 Elashoff, D .; Park NH. ; Wong, DT .; Clinical Cancer Research 2004 10, 8442-8450. Grate, D .; Wilson, C .; Proc. Natl. Acad. Sci. 1999, 96, 6131-6136. Stojanovic, MN .; Kolpashchikov, DM .; J. AM. CHEM. SOC. 2004, 126, 9266-9270. Babendure, J.R .; Adams, SR .; Tsein RY .; J. Am. Chem. Soc. 2003, 125, 14716-14717. Mahara, A .; Iwase, R .; Sakamoto, T .; Yamaoka, T .; Yamana, K .; Murakami, A .; Bioorg. Med. Chem. 2003, 11, 2783-2790. Moriyama, K .; Kimoto, M .; Mistui, T .; Yokoyama, S .; Hirao, I .; Nucleic Acids Res. 2005 33, e129.

  The present invention pays attention to various functions including information processing functions of nucleic acid molecules that are biopolymers, and easily determines and classifies and diagnoses gene expression patterns specific to a specific state (for example, disease or disorder). It aims at providing the technique and means which can be performed.

  As a result of intensive studies to achieve the above object, the present inventor has developed a nucleic acid probe (random DNA probe) comprising a sense sequence encoding a reporter molecule for detection downstream of a promoter sense sequence and a random sequence of about 20 bases. A nucleic acid having a sequence complementary to a sense sequence encoding a promoter nucleic acid sequence and a reporter molecule for detection, and a memory nucleic acid probe storing the sequence information of specific DNA / RNA and storing the sequence information It has been found that a target DNA / RNA present in a sample can be detected and classified using a molecule, and the present invention has been completed.

That is, this invention is the following 1-4.
1. The following steps:
(A) contacting a nucleic acid probe comprising a promoter sense sequence and a random sequence consisting of 5 to 100,000 bases with a sample nucleic acid to form a double strand between the random sequence of the nucleic acid probe and the sample nucleic acid;
(B) decomposing one strand of the nucleic acid probe from the 3 ′ end of the nucleic acid probe to a portion where the nucleic acid probe and the sample nucleic acid form a double strand;
(C) extending a sequence complementary to the sequence of the sample nucleic acid from the portion where the nucleic acid probe and the sample nucleic acid form a double strand to the 3 ′ end side of the nucleic acid probe using the sample nucleic acid as a template;
(D) removing the sample nucleic acid and recovering a nucleic acid probe containing a sequence complementary to the sequence of the sample nucleic acid;
A method for producing a nucleic acid probe having sequence information of a sample nucleic acid.

  In the above method, the nucleic acid is DNA or RNA, although not limited thereto. Specifically, for example, the nucleic acid probe is DNA, and the sample nucleic acid is RNA. The sample nucleic acid is not limited, but a body fluid (saliva, blood, urine, etc.), a cell or tissue, or a nucleic acid collected from these can be used.

  In the above method, as the promoter sequence, for example, a T7 promoter sequence, a T3 promoter sequence, an SP6 promoter sequence, and a tandem sequence obtained by linking these sequences can be used.

  In the above method, the single-strand degradation of step (b) can be performed by, for example, exonuclease I. Further, the extension of the sequence in step (c) can be performed by reverse transcription or replication.

  In the above method, the nucleic acid probe may further contain a sense sequence encoding a detection reporter molecule downstream of the promoter sense sequence. The detection reporter molecule is preferably an RNA molecule that is easy to detect, such as a malachite green aptamer RNA molecule.

  Further, in the above method, a plurality of nucleic acid probes having sequence information of each sample nucleic acid can be prepared using a plurality of sample nucleic acids. A sample nucleic acid whose sequence is not specified can be used.

2. The following steps:
(A) contacting a sample with a nucleic acid probe comprising a promoter sense sequence and a sequence complementary to all or part of the target nucleic acid, and forming a double strand between the nucleic acid probe and the target nucleic acid contained in the sample Forming step,
(B) a step of decomposing a single strand of the nucleic acid probe from the 3 ′ end of the nucleic acid probe, wherein when the nucleic acid probe and the target nucleic acid form a double strand, Decomposing the nucleic acid probe to a part forming a chain;
(C) a detection antisense nucleic acid comprising a sequence complementary to the promoter sense sequence and an antisense sequence encoding a detection reporter molecule is added, and a double strand is formed between the promoter sense sequence and the complementary sequence Forming steps,
(D) adding a polymerase to transcribe an antisense sequence encoding the detection reporter molecule to form a detection reporter molecule;
(E) confirming the transcription reaction in step (d) and detecting a target nucleic acid in the sample;
A method for detecting a target nucleic acid in a sample, comprising:

  In the above method, the nucleic acid probe used in step (a) is prepared by, for example, the method described in item 1 above.

  In the above method, the sample is not limited, but body fluids (saliva, blood, urine, etc.), cells or tissues, or nucleic acids collected from these can be used.

  The nucleic acid is not limited, but DNA or RNA can be used. Specifically, for example, the nucleic acid probe is DNA, and the target nucleic acid is RNA.

  In the above method, as the promoter sequence, for example, a T7 promoter sequence, a T3 promoter sequence, an SP6 promoter sequence, and a tandem sequence obtained by linking these sequences are used. In that case, as the polymerase in step (c), T7 RNA polymerase, T3 RNA polymerase, or SP6 RNA polymerase can be used depending on the promoter sequence.

  In the above method, the single-strand degradation of step (b) can be performed by, for example, exonuclease I.

  Further, as the detection reporter molecule, for example, a malachite green aptamer RNA molecule that is an RNA aptamer molecule that enables fluorescence observation by binding to a fluorescent reagent can be used. In this case, as the detection reagent, malachite green Is used.

  In the above method, the nucleic acid probe may further comprise a sense sequence encoding a detection reporter molecule downstream of the promoter sense sequence, whereby in step (c) the sense sequence encoding the detection reporter molecule and A double strand is formed with an antisense sequence encoding a reporter molecule for detection in the antisense nucleic acid for detection.

  Further, in the above method, a target nucleic acid whose sequence is not specified can be used.

3. A target nucleic acid comprising a nucleic acid probe comprising a promoter sense sequence and a random sequence comprising 5 to 100,000 bases or a nucleic acid probe comprising a promoter sense sequence and a sequence complementary to all or part of the target nucleic acid is detected. Kit for.

  In the above kit, the random sequence is preferably composed of 10 to 1000 bases. The nucleic acid molecule is preferably DNA or RNA.

  In the above kit, the promoter sequence of the nucleic acid probe is not limited, but includes a T7 promoter sequence, a T3 promoter sequence, an SP6 promoter sequence, and a tandem sequence in which these sequences are linked. The nucleic acid probe may further comprise a sense sequence encoding a detection reporter molecule downstream of the promoter sequence.

In the above kit, the promoter antisense sequence part forms a double strand with the promoter sense sequence of the nucleic acid probe, and is a sequence complementary to the promoter sense sequence designed so that the detection reporter molecule encoded by itself can be transcribed. And a nucleic acid comprising an antisense sequence encoding a reporter molecule for detection. The antisense sequence encoding the detection reporter molecule is preferably capable of forming an RNA molecule that is easy to detect, and is a malachite green aptamer RNA molecule that can measure fluorescence by binding to malachite green, which is a fluorescent reagent. Can be used.

  Further, the kit may further include at least one selected from the group consisting of exonuclease I, probe extension enzyme (reverse transcription or replication), RNA polymerase, substrate and detection reagent.

4). The following steps:
(A) producing a memory nucleic acid probe having nucleic acid sequence information derived from a reference subject using the method according to item 1 above;
(B) bringing the memory nucleic acid probe into contact with a nucleic acid derived from a subject exhibiting a specific state or characteristic, and forming a double strand with a nucleic acid having the same sequence information as that of the memory nucleic acid probe;
(C) obtaining a nucleic acid group specific to a subject exhibiting a specific state or characteristic by removing the memory nucleic acid probe;
A method for producing a library of genes specific to a subject exhibiting a specific state or characteristic, comprising:

  In the above method, as a reference subject, for example, a healthy or normal subject can be used. Specific conditions include, for example, diseases or disorders. Moreover, the nucleic acid derived from the reference subject and the nucleic acid derived from the subject exhibiting a specific state or characteristic are, for example, mRNA.

  In the above method, further, (d) a sequence unique to the subject exhibiting the specific condition or characteristic using the method described in the preceding item 1 from the nucleic acid group specific to the subject exhibiting the specific condition or characteristic. It is also possible to prepare a memory nucleic acid probe library having sequence information of genes specific to a subject exhibiting a specific state or characteristic by performing a step of creating a memory nucleic acid probe having information.

  According to the method of the present invention, sequence information of an arbitrary nucleic acid can be stored on a nucleic acid molecule (nucleic acid probe) even if the sequence is not specified. This is effective in efficiently classifying the presence pattern of the target nucleic acid from a huge amount of data including noise. This facilitates a comprehensive search for biomarkers effective for disease diagnosis, and enables the early detection by genetic diagnosis and the expansion of disease regions for which early treatment is effective.

  Moreover, the target nucleic acid in a sample can be detected simply and with high sensitivity using the sequence information stored as described above. Furthermore, based on the nucleic acid patterns comprehensively classified by the method of the present invention, it is possible to easily diagnose a subject or a population of subjects exhibiting a target nucleic acid pattern using various memory nucleic acid probe libraries. It is expected to have a great effect in the medical field.

  In the present invention, specific sequence information is stored in a nucleic acid molecule (random nucleic acid probe) having a function of storing sequence information and a function of inducing luminescence (memory nucleic acid probe). A target nucleic acid having specific sequence information using a nucleic acid molecule (detection antisense nucleic acid) having a function of forming a partial complementary strand and enabling transcription of a reporter molecule for detection, or specific sequence information The target nucleic acid having is removed. The random nucleic acid probe of the present invention can store sequence information whose sequence is not specified, and can store a plurality of sequence information in a plurality of random nucleic acid probes in the same reaction system.

1. Random Nucleic Acid Probe One of the nucleic acid molecules used in the present invention is a random nucleic acid probe. This random nucleic acid probe is a nucleic acid molecule serving as a base for storing sequence information, and is composed of a promoter sense sequence and a random sequence (FIG. 1A). The nucleic acid molecule may further include a sense sequence encoding a reporter molecule for detection that is particularly useful in the detection of the target nucleic acid described later (FIG. 1A). This is not an essential component, but is expected to stabilize the transcription reaction of the reporter molecule for detection described later.

  Random nucleic acid probes can be used for DNA, RNA, DNA / RNA hybrid nucleic acids, peptide nucleic acids (PNA), etc., as long as these sequences contain these sequences so that these sequences can exhibit a sequence information storage function and a detection function utilizing a transcription reaction. Either may be sufficient. Preferably, the random nucleic acid probe is DNA.

  A promoter sequence is a sequence capable of initiating transcription by a polymerase, and specific sequences are known to those skilled in the art. For example, but not limited to, a sequence capable of initiating transcription by RNA polymerase (eg, T7 promoter sequence (SEQ ID NO: 1), T3 promoter sequence (SEQ ID NO: 10), SP6 promoter sequence (SEQ ID NO: 11)) And tandem sequences obtained by linking these sequences. Preferably, SEQ ID NO: 16 is used which is a customized T7 promoter sequence (SEQ ID NO: 1) capable of initiating transcription from DNA to RNA in the presence of T7 RNA polymerase.

  The random sequence can be any sequence as long as it is a random single-stranded nucleic acid sequence of about 5 to 100,000 bases. As will be described later, the random sequence stores the sequence information of the sample nucleic acid by annealing with the sample nucleic acid and performing several processes. Here, according to Non-Patent Document 9, it has been confirmed that the random sequence portion stores the sequence information of the genome of Escherichia coli of about 5 Mbp with 20 bases and can be read based on the stored sequence information. Accordingly, the random sequence has a length of 5 to 100,000 bases, preferably 10 to 1000 bases, more preferably about 10 to 100 bases. Random sequences can be easily prepared by those skilled in the art using known methods. For example, using a DNA synthesizer, a nucleotide with a protecting group is added to a deprotected nucleotide as a substrate. Since a protective group is attached, no further nucleotides are added thereafter, and the desired sequence is synthesized by repeating the addition of one base → deprotection. A random sequence can be easily synthesized by randomly determining the base to be added.

  In the preparation of the memory DNA probe described later, the amount of the random DNA probe used to obtain 1 pmol of a purified memory DNA probe having a length of 100 bases or more using about 10 pmol of the sample RNA was about 100 pmol. Here, the random DNA probe is used in a large excess relative to the sample RNA. Since each random DNA probe has a different random sequence, this is to ensure the diversity of random DNA probes that react with RNA. When creating a random sequence, it should be noted that the sequence should be a random sequence without bias so that various types of target nucleic acid sequence information can be stored. As a precaution for reading, the purified memory nucleic acid probe is considered to react 1: 1 with the target nucleic acid, so it is not necessary to add more memory nucleic acid probe than the target nucleic acid amount. When the concentration is low, the reaction probability decreases, and it is expected that the reaction time can be shortened and the reaction efficiency can be increased by introducing an excessive amount of the memory nucleic acid probe.

Optionally, the sense sequence encoding the detection reporter molecule contained in the random nucleic acid probe can form a double strand with the antisense sequence encoding the detection reporter molecule contained in the detection antisense nucleic acid in the reaction described below. Design as follows. If the promoter sequence part forms a double strand regardless of whether the memory nucleic acid probe contains a sense sequence encoding the detection reporter molecule, RNA polymerase encodes the detection reporter molecule that encodes the detection reporter molecule. The sense sequence is transcribed to become a reporter molecule for detection. Therefore, by detecting the transcription reaction, it can be seen that the promoter sequence portion has formed a double strand, and the promoter sequence portion of the nucleic acid probe on the sense side is present in the reaction solution in a state where it can bind to the antisense nucleic acid for detection. Can be confirmed.

  As a sequence encoding the detection reporter molecule, any sequence known to those skilled in the art can be used. For example, a malachite green aptamer molecule having a function of amplifying malachite green fluorescence by binding to malachite green, which is an organic dye, or an adenosine triphosphate (ATP) aptamer molecule or a flavin mononucleotide (FMN) aptamer improved in function A molecule, a sulforhodamine B aptamer molecule having a function of amplifying the fluorescence of sulforhodamine B, an aptamer having a binding activity with a substance such as a protein, and the like, a biotinylated substrate (yTP) (Non-patent Document 11) is incorporated. Using an avidin-biotin bond to an avidin sensor chip of a protein interaction measuring apparatus (for example, BIACORE) using a crystal oscillator or surface plasmon resonance, the aptamer is immobilized, and the binding activity between the protein and the aptamer is measured. This Examples include sequences encoding biotinylated aptamer molecules that can determine the presence or absence of transcription, RNA molecules that can be observed with fluorescence by binding to 2'-O-methyl RNA introduced with organic electroluminescent element pyrene, and the like. It is done. In addition, if a functional RNA molecule capable of observing that a transcription reaction has occurred by emitting fluorescence at the same time as transcription is developed, it can be used. In addition to this method, for example, RNA can be detected by measuring radiation by incorporating a substrate labeled with a radioactive substance by a method known to those skilled in the art.

  Preferably, as a sequence encoding a reporter molecule for detection, a DNA sequence capable of forming malachite green aptamer RNA (SEQ ID NO: 3, 5, etc.) is used because the detection procedure is simple. To do. Examples of the DNA sequence encoding the malachite green aptamer RNA molecule include, but are not limited to, SEQ ID NOs: 2, 4 and the like (sense sequence) (Non-Patent Documents 7 to 9). It is also possible to use a DNA sequence (SEQ ID NO: 6) (sense sequence) encoding a sulforhodamine B aptamer RNA molecule (SEQ ID NO: 7). Further, for example, a DNA sequence (SEQ ID NO: 18) (sense sequence) (Non-patent Document 11) capable of forming a biotinylated aptamer molecule (SEQ ID NO: 17) (Non-patent Document 11) capable of binding to Raf-1 protein is used. It is also possible to do. Furthermore, an RNA detection reagent such as 2'-O-methyl RNA, which is a fluorescent nucleic acid probe that specifically detects RNA, can be used. In this case, it is possible to use a sequence (sense sequence) such as a sequence (SEQ ID NO: 12) (Non-patent Document 10) that can transcribe RNA that reacts with 2'-O-methyl RNA. In the present invention, it is preferable to use a DNA sequence (SEQ ID NO: 2) (sense sequence) encoding a malachite green aptamer RNA molecule.

  The random nucleic acid probe of the present invention comprises a promoter sense sequence and a random sequence in this order from 5 'to 3'. The sense sequence encoding the detection reporter molecule is included downstream of the promoter sense sequence, ie, between the promoter sense sequence and the random sequence. These sequences may be linked directly or via an appropriate linker.

The last 3 bases (3 'side) of the promoter sense sequence should be designed to overlap the first 3 bases (5' side) of the sense sequence encoding the reporter molecule for detection in order to perform a stable transcription reaction. Is preferred. This is because even if the random nucleic acid probe does not contain a sense sequence encoding a reporter molecule for detection, it forms a double strand with the antisense nucleic acid for detection and performs a stable transcription reaction in the reaction described later. Used for. For example, the 3 bases (GGA) beginning with G at the end of the T7 promoter sense sequence in the random nucleic acid probe prepared in Example 1 are transcribed after formation of a double strand with a detection antisense nucleic acid described later. This is the part. The sequence including these three bases forms a double strand with the antisense nucleic acid for detection, and is important for initiating transcription of the sequence after G by RNA polymerase. Become part of the molecule. The base sequence added at the end of the promoter sequence for performing the stable transcription reaction used here may be any sequence that does not affect the function of the reporter molecule for detection, and is actually prepared in Example 1. The sense strand and antisense strand are designed so that the bases after G form a double strand of 3 or more bases including G from the beginning of the promoter sequence, and preferably at least 3 bases form a double strand. If possible, the number of bases and base species are not limited since stable transcription can be performed even with a single strand thereafter.

  In addition, the random nucleic acid probe of the present invention is a nucleic acid probe in which a complementary strand is formed in a portion of a sense sequence encoding a reporter molecule for detection in a molecule transcribed in a reaction described later, and the RNA molecule transcribed at the end of transcription is transferred. May be incorporated so that the transcription start part and the transcription end part become complementary sequences (for example, the first 3 bases of malachite green aptamer transcribed are GGA and the last 3 bases are Although it is UCC, this part is a complementary strand, and after transcription, annealing with GC, GC, and AU, respectively, facilitates separation from the template. Any combination that forms a chain and does not interfere with the function of the aptamer), communication to amplify fluorescence intensity Connected to the joule (the connecting part of the second module and the connection arrangement designed so as not to prevent the first module and the second module from creating the coupling pockets. Designed according to the first module and the second module). It is known that the fluorescence intensity is amplified by connecting a second module (for example, in the case of malachite green aptamer, a module serving as a binding pocket for theophylline or ATP or flavin mononucleotide (FMN) via a communication module). ) May be incorporated (SEQ ID NOs: 13 to 15) (Non-patent Document 8), or an intermolecular and intramolecularly undesirable secondary between a sense sequence encoding a detection reporter molecule and a random sequence. It may incorporate sequences for preventing to form a granulation.

  Furthermore, at the beginning (5 ′ side) of the promoter sense sequence of the random nucleic acid probe prepared in Example 1, a 4-base sequence “just before the sequence known as the minimum sequence necessary for the T7 promoter to function” AGCT "is included. This sequence is an added sequence since it has generally been reported that the addition of several bases immediately upstream of the T7 promoter binding site helps transcription initiation by T7 RNA polymerase and the amount of transcription is improved. The sequence of these several bases is not particularly limited. When a T7 promoter sequence is selected as the promoter sequence of the random nucleic acid probe, it is preferable to add such arbitrary several bases in order to promote the function of the T7 promoter. The base that can be added is not particularly limited as long as it can promote the function of the T7 promoter, and is about 1 to 5 bases, and the number of bases and the base species are not limited.

2. Memory Nucleic Acid Probe Another nucleic acid molecule used in the present invention is a memory nucleic acid probe, which is a nucleic acid molecule in which specific sequence information is stored. The memory nucleic acid probe is composed of a promoter sense sequence and a sequence (memory portion) in which sequence information is stored, and can be prepared, for example, as shown in FIG. 1A. In addition, the memory nucleic acid probe may include a sense sequence encoding a reporter molecule for detection that is particularly useful in the detection of a target nucleic acid described later (FIG. 1A). This is not an essential component, but is expected to stabilize the transcription reaction of the reporter molecule for detection described later.

  First, a random nucleic acid probe is brought into contact with a sample nucleic acid having sequence information to be stored (a and b in FIG. 1A). The sample nucleic acid may be any of DNA, RNA, DNA / RNA hybrid nucleic acid, peptide nucleic acid (PNA) and the like as long as sequence information can be stored. In addition, by performing an extension reaction using a sample nucleic acid as a template, the sequence of the sample nucleic acid to be used can be not only specified but also unspecified.

The reaction conditions are conditions that allow the random sequence in the random nucleic acid probe and the sample nucleic acid to form a double strand based on their complementarity, and such conditions are known to those skilled in the art. However, it is not necessary for the random sequence and the sample nucleic acid sequence to form a double strand based on perfect complementarity, and it does not leave during subsequent cleavage by 3 ′ → 5 ′ exonuclease, reverse transcription, or extension by replication. Any degree of affinity is sufficient, and a double strand may be formed based on partial complementarity. For example, if a single base forms a double strand, a probe extension reaction using a sample nucleic acid as a template can be performed. When the sample nucleic acid is RNA, the reaction solution is preferably set up on ice in order to avoid incomplete synthesis of the memory nucleic acid probe by RNA degradation and successive reactions. At this time, it is not necessary to specifically denature and anneal RNA, but in the case of RNA having a complicated secondary structure, a denaturation step may be required, and in that case, it is dissolved in nuclease-free water. It is preferable to perform an operation of placing a reaction tube on ice immediately after incubation of the RNA at 65 ° C. to 70 ° C. for 5 minutes to 10 minutes. More preferably, incubation is performed at 70 ° C. for 10 minutes. It is preferable not to denature RNA in a reverse transcription solution. Furthermore, since the random nucleic acid probe has 100 bases or more, before contact with the sample nucleic acid, a 30-second incubation at 94 ° C. followed by rapid cooling on ice is performed to destroy the formation of higher-order structures in the molecule. Perform the operations to be performed as necessary. The sample is prepared so that the concentration of the random nucleic acid probe during the exonuclease reaction is, for example, 50 to 150 mg / L, preferably about 100 mg / L, and the final concentration in the reverse transcription reaction solution is 2 to 3 mg / L.

Subsequently, the nucleic acid that does not form a double strand from the 3 ′ end of the random nucleic acid probe is degraded (c in FIG. 1A). At this time, the sequence of the 3 ′ end side of the random nucleic acid probe is decomposed to the part where the sample nucleic acid is double-stranded. The decomposition method is not particularly limited as long as the single-stranded nucleic acid sequence can be decomposed in the 3 ′ to 5 ′ direction. For example, 3 ′ → 5 ′ exonuclease I having an activity of sequentially decomposing 3 ′, 5′-phosphodiester bonds in the 5 ′ direction from the 3′-OH end of single-stranded DNA can be used. Examples of enzymes having 3 ′ → 5 ′ exonuclease activity include exonuclease I and KOD DN.
A polymerase, T4 DNA polymerase, etc. are mentioned. In the present invention, exonuclease I is preferably used. The specificity of exonuclease I for single-stranded DNA is high, and double-stranded DNA and RNA do not degrade. Since exonuclease I is inactivated by heat treatment at 80 ° C. for 15 minutes, no further chemical treatment is required to stop the reaction.

Next, the probe is extended from the 3 ′ end of the random nucleic acid probe using the sample nucleic acid as a template (d in FIG. 1A). For example, when the random nucleic acid probe is DNA and the sample nucleic acid is RNA, the reverse transcriptase is used and the 5 ′ end side of the sample RNA from the portion where the random DNA probe and the sample RNA form a double strand. Using a single strand as a template, the DNA probe is extended to synthesize a sequence complementary to the sample RNA (reverse transcription). The reverse transcriptase used at this time is not limited as long as it can perform reverse transcription. In addition, when the random nucleic acid probe is DNA and the sample nucleic acid is DNA, DNA replication enzyme (DNA polymerase) is used to remove sample DNA from the portion where the random DNA probe and the sample DNA form a double strand. 'Using the single strand at the end as a template, the DNA probe is extended to synthesize a sequence complementary to the sample DNA (replication). On the other hand, for example, when the random nucleic acid probe is RNA and the sample nucleic acid is DNA, the extension by the enzyme is difficult, so the sequence of the sample nucleic acid is determined and synthesized using a nucleic acid synthesizer to obtain a random probe having a memory portion. It is preferable to produce it. The newly synthesized sequence has a memory portion in which the sequence information of the sample nucleic acid is stored (FIG. 1A).

In the present invention, a nucleic acid probe in which sequence information is stored as described above is referred to as a “memory nucleic acid probe”. In order to recover the memory nucleic acid probe, the sample nucleic acid that forms a double strand with the memory nucleic acid probe is removed (e in FIG. 1A). When the memory nucleic acid probe is DNA and the sample nucleic acid is RNA, the sample RNA is removed using an enzyme that recognizes the DNA-RNA hybrid and degrades the RNA, and the memory DNA probe is recovered. The enzyme that specifically recognizes and degrades such RNA is not particularly limited . Examples include E. coli ribonuclease H and Tth ribonuclease H. Since the deactivation operation is easy, E.I. E. coli ribonuclease H is used. Further, when the memory nucleic acid probe is DNA and the sample nucleic acid is DNA, any method can be used as long as it is a method capable of separating the DNA double strand. Specifically, for example, after a random DNA probe is biotinized in advance and the sample DNA is separated by denaturing the double strand formed by the random DNA probe forming the memory and the sample DNA into single strands. Only the memory DNA probe can be easily recovered using avidin beads or the like. Similarly, when the memory nucleic acid probe is RNA and the sample nucleic acid is DNA, the random nucleic acid probe is biotinylated, and the RNA probe that forms the memory is combined with the sample RNA in a single strand. The memory nucleic acid probe can be easily obtained by a method such as recovering only the memory RNA probe using avidin beads or the like after the sample RNA is cleaved by denaturation into a main strand.

  In addition, the memory nucleic acid probe includes a memory part that stores known sequence information, and a nucleic acid molecule that includes a promoter sense sequence and a memory part, or optionally further includes a sense sequence that encodes a reporter molecule for detection. May be made by synthesis by any method known in the art.

  In the present invention, using a plurality of sample nucleic acids, the plurality of sequence information can be stored in a memory nucleic acid probe in the same reaction system. The sample nucleic acid may be a plurality of types of sample DNA or sample RNA, or a combination of sample DNA and sample RNA. Further, the sequence information of the sample nucleic acid may be specified or specified for all or part of the sample nucleic acid when a memory is created using a random sequence regardless of synthesis. It may not be.

  For example, genomic DNA or mRNA is collected from a reference subject or a subject exhibiting a specific state or characteristic, the genomic DNA or mRNA is used as a sample nucleic acid, and sequence information is stored in a random nucleic acid probe. A memory nucleic acid probe library in which sequence information of a subject is stored, or a memory nucleic acid probe library in which sequence information of a subject exhibiting a specific state or characteristic is stored can be prepared (a and c in FIG. 2). . Here, the reference subject is a subject that does not exhibit a specific state or characteristic, and for example, a healthy or normal subject can be used.

Further, when the library is produced, the memory nucleic acid probe can be used for removing a specific nucleic acid (DNA / RNA). For example, when removing a specific DNA, remove the memory nucleic acid probe specifically bound to the specific DNA as a complex by applying biotin modification to the memory DNA nucleic acid probe and recovering with avidin beads. Is possible. In the case of removing specific RNA, in addition to removal by recovery of the memory DNA probe to which the specific RNA is bound, an enzyme that degrades only the RNA specifically bound to the memory DNA probe (for example, E. coli ribonuclease H, RNA can also be selectively removed using Tth ribonuclease H or the like.

  By utilizing the above-described selective exclusion function of the memory nucleic acid probe, for example, a library of genes (for example, cancer marker genes) specific to a specific state or characteristic can be efficiently produced. For example, by inserting a memory nucleic acid probe library in which the sequence information of mRNA of a healthy person is stored in mRNA in saliva of a cancer patient, And then degrading the mRNA hybridized with the memory nucleic acid probe using a degrading enzyme, the mRNA having the same sequence as that of a healthy person can be selectively excluded (b in FIG. 2). That is, mRNA specific to cancer patients can be collected. A memory nucleic acid probe library in which sequence information specific to a cancer patient is stored can be prepared by using the mRNA specific to the cancer patient collected as a sample nucleic acid (c in FIG. 2). Further, by determining the sequence of a memory nucleic acid probe library in which sequence information specific to cancer patients is stored, it is possible to clarify the sequence information of genes specific to cancer. The method of the present invention makes it possible to create a memory nucleic acid probe by making use of hybridization in a random sequence portion of a random nucleic acid probe, even if the target nucleic acid sequence is unknown, and to create a sample nucleic acid. Therefore, it is possible to eliminate sequences that cause unnecessary noise without determining the sequence in advance (that is, to remove the mRNA of healthy subjects from the mRNA of cancer patients). It is effective in conducting an exhaustive search for new biomarkers that can be used for disease diagnosis by analysis. When searching for genes that show abnormal expression in a specific disease, by removing noise that hinders analysis, analysis using a regression tree model, logistic regression model, etc. is facilitated, and significant uniqueness can be identified from a large number of samples. It is possible to easily classify unknown genes that are effective for sex diagnosis, and the efficiency of the gene determination process can be increased.

3. Detection of Target Nucleic Acid Using Memory Nucleic Acid Probe In the present invention, when the memory nucleic acid probe is brought into contact with the sample (f in FIG. 1B), a sequence complementary to the sequence information stored in the memory nucleic acid probe is stored in the sample. A target nucleic acid (that is, a nucleic acid having the same sequence as the whole or a part of the sample nucleic acid used to store the sequence information), a reaction occurs between the target nucleic acid and the memory nucleic acid probe, and By detecting the reaction, the target nucleic acid can be detected in the sample.

  The sample to be reacted with the memory nucleic acid probe is not particularly limited as long as it contains a nucleic acid (genomic DNA, cDNA, mRNA, total RNA, etc.) having sequence information. For example, it can be a body fluid (saliva, blood, urine, etc.), cell, tissue derived from a subject. Preferably, nucleic acid components are purified and / or amplified from these sources and used as samples. In addition, the sample may be derived from nature (sample prepared from a subject, genomic DNA, mRNA, etc.) or a synthesized sample (eg, DNA library, genomic library, etc.). Good. The nucleic acid contained in the sample may be one kind or plural kinds.

When a target nucleic acid having a sequence complementary to the sequence information stored in the memory nucleic acid probe is present in the sample, the memory portion of the memory nucleic acid probe and all or a part of the target nucleic acid are bound (DNA-DNA hybrid). Or DNA-RNA hybridization) (g in FIG. 1B). Here, in the same manner as when the memory nucleic acid probe was prepared, an enzyme having an activity of degrading the nucleic acid from the 3 ′ end to the portion where the memory nucleic acid probe and the target nucleic acid form a double strand, such as exonuclease I, is added. Used to degrade all memory nucleic acid probes that do not form double strands. Memory nucleic acid probes that are not hybridized with the target nucleic acid are almost always degraded (when a complementary strand is formed between molecules or within a molecule, a part remains undegraded). Therefore, when the target nucleic acid is present in the sample, the memory nucleic acid probe remains without being decomposed (h in FIG. 1B), and when the target nucleic acid is not present, almost all of the memory nucleic acid probe is decomposed. become.

  Subsequently, a detection antisense nucleic acid containing a promoter antisense sequence and an antisense sequence encoding a detection reporter molecule is introduced (i in FIG. 1B). The antisense nucleic acid for detection used in the present invention is a nucleic acid molecule necessary for detecting a target nucleic acid in a sample based on a transcription reaction, and is a sequence complementary to a promoter sequence of a random nucleic acid probe or a memory nucleic acid probe. And an antisense sequence encoding a detection reporter molecule. The antisense nucleic acid for detection is an antisense sequence that encodes a reporter molecule for detection, whether or not the random nucleic acid probe or memory nucleic acid probe includes a sense sequence that encodes the reporter molecule for detection. Need to be included. This is not a sense sequence that encodes a reporter molecule for detection in a random nucleic acid probe or a memory nucleic acid probe, but a template for a detection reporter molecule to be transcribed in the fluorescence detection described later. This is because the antisense sequence encodes a reporter molecule for detection in the sense nucleic acid.

  The antisense nucleic acid for detection is DNA, RNA, DNA / RNA hybrid nucleic acid, peptide nucleic acid (as long as it contains these sequences so that it can function to detect a reaction caused by the presence of the target nucleic acid in the sample. PNA) or the like may be used. Preferably, the antisense nucleic acid for detection is DNA.

  The promoter antisense sequence in the antisense nucleic acid for detection is a sequence capable of initiating transcription by a polymerase and is complementary to the promoter sense sequence used in the above-mentioned random nucleic acid probe or memory nucleic acid probe. It is not particularly limited.

  In addition, because a transcription reaction occurs with RNA polymerase using an antisense sequence encoding a detection reporter molecule as a template, a detection reporter molecule is formed, so the transcription reaction is observed by detecting the detection reporter molecule . As will be described later, when a transcription reaction is observed, the memory nucleic acid probe recognizes the target nucleic acid to form a double strand, thereby avoiding cleavage by exonuclease or preventing the transcription reaction by the target nucleic acid. Promoter part between the memory nucleic acid probe and the detection antisense nucleic acid due to the fact that the formation of intermolecular or intramolecular higher order structure with the promoter sequence part is inhibited and the promoter sequence can form a double strand. It shows that the double strand of is formed. If there is no target nucleic acid, all of it is cleaved by exonuclease and there is no memory nucleic acid probe, there is no sense sequence corresponding to the antisense nucleic acid for detection, or there is no target nucleic acid. When the promoter sequence part forms a non-specific higher order structure between or within the promoter sequence part and the promoter part part prevents the detection antisense nucleic acid from forming a double strand, the polymerase The transcription reaction does not occur because the nucleic acid sequence to be transcribed cannot be recognized. If the promoter sequence forms a double strand between the sense sequence and the antisense sequence, transcription by the polymerase occurs, and the antisense sequence encoding the reporter molecule for detection of the antisense nucleic acid is transcribed to perform the detection operation. This indicates that the target nucleic acid molecule having the sequence information stored in the memory nucleic acid probe exists.

As an antisense sequence encoding the detection reporter molecule, any sequence known to those skilled in the art can be used. For example, a malachite green aptamer molecule having a function of amplifying malachite green fluorescence by binding to malachite green, which is an organic dye, or an adenosine triphosphate (ATP) aptamer molecule or a flavin mononucleotide (FMN) aptamer improved in function A molecule, a sulforhodamine B aptamer molecule having a function of amplifying the fluorescence of sulforhodamine B, an aptamer having a binding activity to a substance such as a protein, and the like, and biotinylated substrate (yTP) (Non-patent Document 11) Using an avidin-biotin bond to an avidin sensor chip of a protein interaction measuring apparatus (for example, BIACORE) using a crystal oscillator or surface plasmon resonance, the aptamer is immobilized, and the binding activity between the protein and the aptamer is measured. This Biotinylated aptamer molecules that can determine the presence or absence of transcription by, in combination with 2'-O- methyl-type RNA introduced an organic electroluminescent device pyrene encoding an RNA molecule to amplify the fluorescence sequences.

Preferably, as an antisense sequence encoding a reporter molecule for detection, an antisense sequence of a sequence encoding a DNA whose transcribed sequence can form malachite green aptamer RNA (SEQ ID NO: 3, 5, etc.) is used. To do. Examples of the DNA sequence encoding the malachite green aptamer RNA molecule include, but are not limited to, antisense sequences for SEQ ID NOs: 2, 4 and the like (Non-Patent Documents 7 to 9). It is also possible to use an antisense sequence of a DNA sequence (SEQ ID NO: 6) encoding a sulforhodamine B aptamer RNA molecule (SEQ ID NO: 7). Furthermore, an RNA detection reagent such as 2′-O-methyl RNA, which is a fluorescent nucleic acid probe that specifically detects RNA, can be used. When the RNA detection reagent is used for RNA detection, an antisense sequence such as a sequence capable of transcribing RNA that reacts with 2′-O-methyl RNA (SEQ ID NO: 12) (Non-patent Document 10) is used. It is also possible. In addition, for example, an antisense sequence of a DNA sequence (SEQ ID NO: 18) (Non-patent Document 11) that can form a biotinylated aptamer molecule (SEQ ID NO: 17) (Non-patent Document 11) that can bind to the Raf-1 protein is used. It is also possible to do. In the present invention, it is preferable to use an antisense sequence of a DNA sequence (SEQ ID NO: 2) encoding a malachite green aptamer RNA molecule.

  When the random nucleic acid probe or the memory nucleic acid probe contains a sense sequence encoding a detection reporter molecule, the antisense sequence encoding the detection reporter molecule is complementary to the sense sequence encoding the detection reporter molecule. Select an appropriate sequence so that

  The antisense nucleic acid for detection contains 5 ′ to 3 ′ in the order of an antisense sequence encoding a detection reporter molecule and a sequence complementary to the promoter sense sequence. These sequences may be linked directly or via an appropriate linker. If the random nucleic acid probe or memory nucleic acid probe to be used contains an additional sequence (base) before the promoter sense sequence part or between the promoter sequence part and the random sequence part or memory sequence part, the additional It is preferable to add a sequence or a base as appropriate to the antisense nucleic acid for detection so as to be complementary to the sequence.

  The antisense nucleic acid for detection can be easily designed by those skilled in the art so as to include the above-described components. For example, in the case where a random nucleic acid probe or a memory nucleic acid probe uses a T7 promoter sense sequence shown in SEQ ID NO: 16 and a sense sequence encoding a detection reporter molecule shown in SEQ ID NO: 2, the sequence is used as a detection antisense nucleic acid. The sequence shown in number 9 can be used.

When the memory nucleic acid probe remains without being decomposed in the above decomposition reaction (h in FIG. 1B) (or the presence of the target nucleic acid causes the promoter sequence portion of the memory nucleic acid probe to be free without forming a higher order structure). The detection antisense nucleic acid hybridizes with the promoter sense sequence of the memory nucleic acid probe to form a double strand (
As a result of j) in FIG. 1B, recognition by RNA polymerase becomes possible, and a transcription reaction takes place by RNA polymerase using an antisense sequence encoding a reporter molecule for detection as a template (k in FIG. 1B).

  Therefore, a polymerase (RNA polymerase) that recognizes a double-stranded promoter sequence and initiates transcription is inserted, and a sequence encoding a detection reporter molecule is transcribed to form a detection reporter molecule (k in FIG. 1C). . The polymerase to be used is not particularly limited, and can be appropriately selected according to the promoter sequence. For example, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase or the like can be used, and when a tandem promoter is used, any one of these polymerases can be used. In the above step, when a detection antisense nucleic acid containing a T7 promoter antisense sequence is introduced to form a double strand, a transcription reaction is performed by recognizing the T7 promoter sequence forming the double strand. It is preferable to use T7 RNA polymerase (available from Ambion, etc.).

  Next, the presence of the reporter molecule is observed based on the characteristics of the formed reporter molecule for detection. 1C and 1M show examples of malachite green aptamers in which fluorescence can be observed by binding to malachite green, which is a detection reagent (a substance that induces or amplifies luminescence). The method for observing the reporter molecule differs depending on the type of the reporter molecule for detection. For example, malachite green aptamer is used for malachite green aptamer, sulforhodamine B is used for sulforhodamine B aptamer, and organic electroluminescence is used for OMUpy aptamer. 2'-O-methyl RNA into which element pyrene is introduced. In the case of observing fluorescence, the fluorescence can be excited with a wavelength suitable for the type of reagent for fluorescence detection, and can be performed using, for example, a fluorescence microscope. Molecules that fluoresce themselves when transcribed have not been developed yet, but if such molecules are developed, such molecules can also be used as detection reporter molecules.

  The confirmation of the transcription reaction of the reporter molecule for detection may not be based on fluorescence observation. For example, a biotinylated aptamer molecule obtained by incorporating a biotinylated substrate into an aptamer having a binding property with a protein or the like is applied to an avidin sensor chip of an interaction measuring device (for example, BIACORE) using a crystal oscillator or surface plasmon resonance. The transcription reaction can be quantitatively and qualitatively determined by measuring the binding activity between an aptamer and a protein by immobilizing with an avidin-biotin bond.

  When it is confirmed that the reporter molecule for detection is transcribed, it can be determined that the target nucleic acid is contained in the sample. In the present invention, “detection” means not only determining the presence of a target nucleic acid but also quantifying the target nucleic acid. The amount of the target nucleic acid can be determined, for example, by comparing the fluorescence intensity with the control.

The method of the present invention can be used to determine or predict the presence or risk of a subject's disease. This determination is made, for example, by a memory nucleic acid probe library in which sequence information of healthy or normal subjects is stored as described above, and a memory nucleic acid probe library in which sequence information of subjects suffering from a specific disease is stored (see FIG. 2) is used to detect the presence or absence of a disease specific nucleic acid in a sample of a subject (FIGS. 3A and B). The memory nucleic acid probe is preferably immobilized on the microarray or microchip by techniques known in the art (FIG. 3A). The sample of the subject is not limited, but bodily fluids such as saliva, blood and urine, cells and tissues, nucleic acids purified from these, and the like can be used. In particular, from the viewpoint of easy collection, it is preferable to use saliva or a nucleic acid purified from saliva as a sample. It is known that there are about 3000 types of mRNA in saliva, of which about 280 are specific for healthy individuals. It has also been reported that 1679 genes contained in the saliva of oral cancer patients show different expression from that of healthy individuals, and in particular, 7 kinds of mRNAs are increased about 3.5 times in cancer patients. The diagnosis of diseases using the mRNA in them as a biomarker has already been put into practical use in the diagnosis of these two diseases.

  The above-described method of the present invention can be performed more easily by using a kit. Such kits include random nucleic acid probes or memory nucleic acid probes (nucleic acid molecules), and antisense nucleic acids for detection, as well as enzymes and reagents required for the reaction, such as exonuclease I, reverse transcriptase, polymerase, substrate and required detection. Reagents are included. The kit may further contain ribonuclease H, a buffer, and the like.

  Moreover, DNA / RNA can be classified using the method of the present invention. The classification is performed for the purpose of arranging, for example, a group of samples showing the same or similar RNA expression pattern based on the pattern. Specifically, a method for classifying a sample group showing the same or similar RNA expression pattern can be performed as follows.

Create a memory nucleic acid probe library that stores the expressed RNA from a sample group showing a specific RNA expression pattern, and comprehensively investigate which gene the memory nucleic acid probe reacts with using a cDNA array. A dedicated cDNA array containing only the memory nucleic acid probe prepared is prepared. Next, a plurality of samples whose expression patterns have not been analyzed are prepared and analyzed using the prepared dedicated cDNA array. If luminescence occurs in all or most of the reactions, it can be said that the sample has the same or similar expression pattern. The reaction of the memory nucleic acid probe on the DNA array can be observed by labeling the memory nucleic acid probe as a specimen using a label such as an ARES ^™ DNA labeling kit (Invitrogen). Alternatively, when the reporter molecule for detection has an observation function by fluorescence, it is simple and easy to introduce the necessary reagents such as the antisense nucleic acid for detection, T7 polymerase, and the detection reagent without performing such labeling. The reaction can be easily observed. In addition, RNA labeling for observation on an array of sample RNA can be easily performed using a known method known to those skilled in the art, such as ULYSIS (registered trademark) direct labeling kit (Invitrogen). . When the memory nucleic acid probe and the detection kit having a fluorescence observation function such as malachite green aptamer are used as the detection reporter molecule of the present invention on the array itself, there is no need to perform such labeling, and the fluorescence of the memory nucleic acid probe A detection function can be used.

  In addition, by using the library prepared using the method of the present invention as described above, RNA that is not an indicator from the sample group (that is, not specific to the sample group) is removed in advance by subtraction as noise. Thus, it is possible to improve the efficiency of classification of RNA patterns characteristic to the above. For example, when classifying an expression pattern characteristic of a disease, using a library prepared using a sample from a healthy person, the pattern that is also present in the expression pattern of a healthy person stored in the library in advance is excluded. Classification can be performed.

Moreover, the sample which shows a similar expression pattern can be searched using the method of this invention. Here, a sample showing a similar expression pattern is, for example, when the target nucleic acid is RNA and the type of RNA whose expression level is increasing is examined, quantitative expression of RNA in the total RNA sample Samples that are similar in the combination of RNAs that have undergone a quantity check and whose expression levels are increasing are similar to each other, and organisms from which samples having such a similar expression pattern are collected show the same or similar state, that is, the same You may be sick or have a similar health condition. In other words, by examining the similarities in the RNA expression pattern of samples, if it is collected from a group of patients with a common disease, it is also possible to search for RNA combinations as disease markers for the disease In addition, if there is a combination of RNA as a disease marker that has been clarified in advance, it can be used for early detection of the disease of the organism from which the sample was collected. The advantage of the present invention is that classification can be made more efficient by performing subtraction of nucleic acid molecules that cause noise before the sequence is specified in advance, and the process of fluorescent labeling of the sample is unnecessary. It is possible to quantitatively amplify RNA in a sample using a transcription reaction, and to detect using a characteristic that a target nucleic acid protects from higher-order structure formation of a promoter sequence part by using a transcription reaction. For example, the sensitivity can be improved. Even if a detection reporter sequence is not incorporated into the detection antisense nucleic acid, a method known to those skilled in the art (for example, by directly fluorescently labeling the detection antisense nucleic acid and using the fluorescence to detect the memory nucleic acid probe). ), It is possible to determine whether the memory nucleic acid probe remains, that is, the presence of the sample nucleic acid. In that case, quantitative amplification that is possible when using a transcription reaction as in the present invention cannot be performed, but it is not necessary to perform a transcription reaction with a polymerase, which is convenient.

  Furthermore, the present invention incorporates not only a diagnostic / classification system but also a nucleic acid sequence encoding a drug (for example, a nucleic acid sequence encoding an antisense RNA, a double-stranded RNA, etc. known as an RNA drug) downstream of a promoter sequence. Thus, a drug that senses the presence of a specific target nucleic acid and releases the drug can be produced and used to treat or prevent a disease or disorder.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.

Production of Random DNA Probe In this example, a random DNA probe serving as a basis for storing nucleic acid sequence information was produced. Specifically, as a model DNA of this DNA probe, a 79-base sense DNA containing a sequence complementary to the malachite green aptamer sequence and a 20-base random sequence downstream of the T7 promoter sense sequence (underlined portion) ( 5′-AGCT TAATACGACTCACTATA GGATCCCGACTGGCGAGAGCCAGGTAACGAATGGATCCNNNNNNNNNNNNNNNNNNNN-3 ′; SEQ ID NO: 8) was synthesized. In addition, a 59-base detection antisense DNA (5′-GGATCCATTCGTTACCTGGCTCTCGCCAGTCGGGATCCTATAGTGAGTCGTATTAAGCT-3 ′; SEQ ID NO: 9) used in Example 4 described later was prepared.

Preparation of sample RNA and target RNA In this example, a sample RNA used for storing sequence information in a random DNA probe and a target RNA for detection using a memory DNA probe were prepared.

A pTRI-β-actin-Mouse DNA control template for control experiments included in the MAXIscript (registered trademark) T7 kit (Ambion) is prepared for the preparation of sample RNA and target RNA. The control template pTRI-β-actin-Mouse was used to generate an antisense mouse β-actin probe and was 250 bp between the restriction enzyme site KpnI-Xba1 of the mouse β-actin gene subcloned from pAL41. It is a fragment and is a plasmid prepared in a linear form, and transcriptionally controlled by SP6, T7 and T3 tandem promoters. When transcribed with SP6, T7 and T3 RNA polymerase, 334 bases and 304
Base and 276 bases in length.

In this example, since the transcription reaction by T7 RNA polymerase is used in the detection of target RNA described later, sample RNA and target RNA are prepared using SP6 RN using MEGAscript (registered trademark) SP6 kit (Ambion).
Performed with A polymerase, purified using RNeasy® Mini (QIAGEN) and quantified with a spectrophotometer (BECKMAN DU640). pTRI-β-actin-Mouse (0.5 mg / mL) was prepared in a volume of 1 μL per 20 μL of the reaction solution and purified by eluting with 30 μL of nuclease-free water using two RNeasy mini columns. The obtained RNA was 1.34 μg / μL and about 60 μL. Dispense and store at -80 ° C until use.

Production of Memory DNA Probe Using Random DNA Probe In this example, the random DNA probe produced in Example 1 was used to store the sequence information of the sample RNA produced in Example 2 in the random DNA probe. (FIG. 1A).

About 2.4 μg of 100 pmol of random DNA probe (Example 1) was added to 1.3 μg of 12 pmol pTRI-β-actin-Mouse sample RNA (Example 2) (a in FIG. 1A), and nuclease-free water The sample was heated to 70 ° C. for 10 minutes in order to break the RNA having a higher-order structure in the molecule. Then, after rapidly cooling to 4 ° C., the sample RNA and the random sequence portion of the random DNA probe were incubated at 30 ° C. for 30 minutes in order to anneal. Here, since the random DNA probe is long, as an operation for destroying the higher-order structure formed in the molecule, the random DNA probe may be incubated at 94 ° C. for 30 seconds before introducing the sample RNA. To 10 μL of this solution, 1.5 μL of 10 × exonuclease I buffer attached to exonuclease I (Takara) and 2 μL of exonuclease I (5 units / μL) were added to make 15 μL with nuclease-free water. Here, one unit of exonuclease I is defined as an enzyme activity that produces 10 nmol of an acid-soluble degradation product in 30 minutes under conditions of 37 ° C. and pH 9.5 using heat-denatured calf thymus DNA as a substrate. Here, 10 units are added, but when 50 units are added per reaction, the cleavage can be performed more completely. Next, the mixture is heated at 37 ° C. for 30 minutes to decompose from the 3 ′ end of the random DNA probe not forming a double strand (c in FIG. 1A). If a double strand is formed, the degradation stops there. Thereafter, in order to inactivate exonuclease I, a heat treatment is performed at 80 ° C. for 15 minutes, followed by rapid cooling to 4 ° C.

Next, reverse transcription is carried out using SuperScript ^™ II (GIBCOBRL) as a template with RNA forming a double strand (d in FIG. 1A). After adding 4 μL of 5 × First Strand buffer attached to SuperScript ^™ II, 2 μL of 1M DTT, 1 μL of 10 mM dNTP mix to the total amount of the reaction solution, and incubating again at 25 ° C. for 10 minutes to promote annealing of random sequences, Incubated for 2 minutes at 42 ° C. Further, 1 μL of SuperScript ^™ II was added and reacted at 42 ° C. for 50 minutes, followed by incubation at 55 ° C. for 15 minutes as a treatment for random sequencing, followed by heating at 70 ° C. for 15 minutes to inactivate SuperScript ^™ II.

To remove the annealed sample RNA (e in FIG. 1A), 2 units of ribonuclease H (Takara) 60 units / μL were added, incubated at 37 ° C. for 20 minutes, and then heated at 65 ° C. for 20 minutes. A deactivation treatment of H was performed. Here, one unit of ribonuclease H is the amount of enzyme necessary to produce 1 nmol acid-soluble ribonucleotide at 37 ° C. for 20 minutes, and 20 mM HEPES-KOH as a reaction solution.
(PH 7.8), 50 mM KCl, 10 mM MgCl ₂ , 1 mM DTT, and 20 μM radiolabeled poly (rA): poly (dT) as a substrate. The memory DNA probe in which the sequence information of the sample RNA of pTRI-β-actin-Mouse was recorded was thus completed.

  Furthermore, only the memory DNA probe of 100 mer or more was purified using the QIAquick PCR Purification kit. Ten times the amount of the reaction solution is extracted with 30 μL of nuclease-free water using two columns, and the concentration is measured with an absorptiometer. About 56 μL of the memory DNA probe of 0.005 μg / μL = ˜0.02 pmol / μL was obtained. Stock at -20 ° C until use. Of 1000 pmol of the random DNA probe input for the production of the memory DNA probe, 1.12 pmol was recovered. If necessary, an unpurified product that is not purified by the QIAquick PCR Purification kit is also prepared.

Detection of Target RNA Using Memory DNA Probe In this example, target RNA was detected using the memory DNA probe in which sequence information was stored in Example 3 (see FIGS. 1B and 4). Specifically, a memory DNA probe for purified pTRI-β-actin-mouse RNA (Example 3)
The detection experiment shown in FIG.

  Purified memory DNA probe 46 μL (0.23 μg) was added (f in FIG. 1B, g in FIG. 4), 1 μL 1.34 μg / μL pTRI-β-actin-Mouse RNA (target RNA, prepared in Example 2) ) Was annealed (g in FIG. 1B, h in FIG. 4). In order to break the RNA having a higher order structure in the molecule, it was heated at 70 ° C. for 10 minutes, rapidly cooled to 4 ° C., and then incubated at 30 ° C. for 30 minutes. Here, since the memory DNA probe is long, an operation for breaking the higher order structure formed in the molecule may be performed by incubating the memory DNA probe for 30 seconds at 94 ° C. before introducing the target RNA.

  Add 5 μL of 10 × exonuclease I buffer attached to exonuclease I and 4 μL of exonuclease I (5 units / μL) to the total amount of this annealing solution to make 56 μL, and heat at 37 ° C. for 30 minutes to form a double strand. Disassembly from the 3 ′ end of the non-memory probe was performed (h in FIG. 1B, i in FIG. 4). If a double strand is formed, the degradation stops there. Thereafter, in order to inactivate exonuclease I, a heat treatment is performed at 80 ° C. for 15 minutes, followed by rapid cooling to 4 ° C. Here, 10 units are added, but when 50 units are added per reaction, the cleavage can be performed more completely.

  Further, 1 μL of detection antisense DNA (prepared in Example 1) was added at 100 pmol (i in FIG. 1B), and the fluorescent aptamer sequence was transferred using the MEGAscript (registered trademark) T7 kit. After incubating at 68 ° C. for 30 minutes, it was annealed with the promoter sequence and malachite green aptamer sequence portion of the memory DNA probe (j in FIG. 1B, j in FIG. 4). In accordance with the instructions of the MEGAscript (registered trademark) T7 kit, ATP, CTP, GTP, and UTP were each added to 12.5 μL, 10 × reaction buffer was added to 12.5 μL, and then the enzyme mix containing T7 polymerase was added in 12. 5 μL was added and incubated at 37 ° C. for 16 hours to carry out a transcription reaction (k in FIG. 1B).

Similarly, as control 1, random DNA probe 0.5 μL (50 pmol)
(F in FIG. 1B, a in FIG. 4), 1.34 μg / μL pTRI-β-actin-
1 μL of Mouse RNA (target DNA, prepared in Example 2) was annealed to 10 μL with nuclease-free water (g in FIG. 1B, b in FIG. 4). In order to break the RNA having a higher order structure in the molecule, it was heated at 70 ° C. for 10 minutes, rapidly cooled to 4 ° C., and then incubated at 30 ° C. for 30 minutes. 2 μL of 10 × exonuclease I buffer attached to exonuclease I and 2 μL of exonuclease I (5 units / μL) are added to the total amount of the annealing solution to make 20 μL with nuclease-free water. Decomposition from the 3 ′ end of the random memory that did not form a double strand by heating at 37 ° C. for 30 minutes was performed (h in FIG. 1B, c in FIG. 4). If a double strand is formed, the degradation stops there. Thereafter, in order to inactivate exonuclease I, heat treatment was performed at 80 ° C. for 15 minutes, followed by rapid cooling to 4 ° C.

  Furthermore, 1 μL of detection antisense DNA (prepared in Example 1) was added at 100 pmol (i in FIG. 1B), incubated at 94 ° C. for 30 seconds and at 68 ° C. for 30 minutes, and the promoter sequence of the memory DNA probe and malachite green It annealed with the aptamer arrangement | sequence part (j of FIG. 1B, d of FIG. 4). Further, the fluorescent aptamer sequence is transcribed using MEGAscript (registered trademark) T7 kit. According to the instructions of the kit, 5 μL each of ATP, CTP, GTP, UTP, 10 μl reaction buffer, 5 μL of the sample, and then 5 μL of enzyme mix containing T7 polymerase were added and incubated at 37 ° C. for 16 hours for transcription. The reaction was performed (k in FIG. 1B).

Similarly, as a control 2, 0.5 μL (50 pmol) of a random DNA probe not cleaved with exonuclease I and 1 μL of detection antisense DNA 1
00 pmol was added, and 20 μL was added with nuclease-free water, followed by annealing at 94 ° C. for 30 seconds and at 68 ° C. for 30 minutes. Fluorescent aptamer sequences were transcribed using MEGAscript® T7 kit. All samples were added to 5 μL each of ATP, CTP, GTP and UTP, 5 μL of 10 × reaction buffer, 5 μL of enzyme mix containing T7 polymerase was added, and incubated at 37 ° C. for 16 hours.

A fluorescent reagent was introduced into these samples to verify the present invention. In the sample where the random / memory DNA probe remains, the detection antisense DNA anneals with the sense portion of the memory DNA probe, and transcription of the malachite green aptamer molecule is initiated by the T7 RNA polymerase included in the MEGAscript (registered trademark) T7 kit. (K in FIG. 1B). 2x observation buffer (200 mM KCL) containing malachite green at the time of observation
Add 10 mM MgCl ₂ , 20 mM HEPES, 20 μM MG [Malachite Green]) in the same volume as the reaction solution (l in FIG. 1C, k in FIG. 4), and observe fluorescence using a plate reader (Molecular Device, SPECTRAmax GEMINI). went. Since malachite green reacts with the aptamer at a theoretical value of 1: 1, 10 μM of malachite green aptamer can be detected at a final concentration of 10 μM of malachite green (m in FIG. 1C, l in FIG. 4). Since production of RNA 80 to 120 times the mass of the input DNA is guaranteed, 10 μM is expected to be about 300 times the amount of aptamer produced by the expected purified memory DNA probe. Conceivable.

  Here, the following quantities are defined in order to compare the transfer efficiency. Random / memory DNA probe reaction when observing the transcription reaction by diluting 20 μL of the transcription reaction solution with an observation buffer using a random / memory DNA probe of 50 pmol and 1.21 μg is defined as 1 unit.

FIGS. 5-6 are the observation results by the plate reader (Molecular device, SPECTRAmax GEMINI) based on an Example. The vertical axis represents the amount of fluorescence when excited at 620 nm, and the horizontal axis represents the observation wavelength. RNA, ExoI, and MG in the figure represent RNA, exonuclease, and malachite green, respectively, and indicate whether the process of adding each with + and-symbols is performed (+) or not (-). First, in order to show that fluorescence cannot be observed unless a fluorescent aptamer molecule and malachite green are present at the same time in FIG. 5, the result of observation with control 2 prepared with a random DNA probe without exonuclease cleavage added with malachite green The results of observation without adding malachite green (control 3) and the results of observation of only the fluorescence of malachite green in the observation buffer are shown (control 4). Only the random DNA probe under the condition of control 3 or only malachite green (MG) of control 4 shows no observable fluorescence on this scale. The fluorescence of the random DNA probe before the memory preparation and malachite green was measured with 5 units of malachite green and 5 units of the random DNA probe before the memory preparation using a total amount of 100 μL, respectively. 5 to 6, the maximum value when fluorescence is observed by adding RNA having sequence information stored in the memory part to the unpurified memory nucleic acid probe before purification by the QIAquick PCR Purification kit shown in FIG. 6. The standardization is performed with 1 being 1.

FIG. 6 shows that an unpurified memory DNA probe and a purified memory DNA probe prepared from 100 pmol random DNA probe, and a 50 pmol random DNA probe (control 1) were all reacted with the target RNA and exonuclease I was cleaved. It is the observation result which experimented.

Here, a comparison between a random DNA probe (control 1) subjected to the reaction with the target RNA of FIGS. 5 and 6 and cleaving with exonuclease I and a random DNA probe not subjected to both (control 2) is as follows. It is inferred. At this time, the reaction with the target RNA was not performed in the control 2, but the control 1 was reacted with the target RNA. Controls 1 and 2 confirmed that the same level of fluorescence was observed. This is considered to indicate the possibility that RNA is preventing the formation of intermolecular and intramolecular complementary strands with the sequence portion of the antisense nucleic acid for detection. If the memory portion of the memory nucleic acid probe has sufficient specificity to form a complementary strand with the target nucleic acid, the promoter sequence portion of the memory nucleic acid probe that anneals the target nucleic acid and the antisense nucleic acid for detection is double-stranded. The formation prevents unwanted intermolecular and intramolecular complementary strands between the memory nucleic acid probes, thereby protecting the antisense nucleic acid for detection from annealing to the sense sequence portion of the memory nucleic acid probe. If sufficient sequence information is not stored in the memory nucleic acid probe or if the target nucleic acid does not exist, the random sequence portion remaining without forming the memory of the memory nucleic acid probe or the memory sequence portion forming the memory An anti-sense for detection is formed by forming a higher-order structure with the promoter sequence part between and within the molecule. Inhibit annealing of the scan nucleic memory nucleic acid probe is considered significantly inhibit fluorescence detection reaction even if considered to be present a sufficient amount. Therefore, only when there is a target nucleic acid having a sequence stored by the memory nucleic acid probe, the promoter antisense sequence of the antisense nucleic acid for detection can form a double strand with the promoter sense sequence portion of the memory nucleic acid probe, It is expected that a reaction necessary for RNA polymerase to recognize a double-stranded promoter sequence and detect fluorescence occurs, and that the target RNA can be detected more effectively by this fluorescence. In other words, the molecular structure of the memory nucleic acid probe itself, which inhibits the formation of a double strand between the promoter sense sequence portion and the promoter antisense sequence of the detection antisense nucleic acid in the absence of the target nucleic acid, increases the specificity of RNA detection. It can be said that Two experiments using a random DNA probe before memory preparation were performed in the same 5 units with a total volume of 100 μL. Fluorescence is detected with a random DNA probe that has been subjected to exonuclease I treatment before memory preparation, because it forms complementary strands within the molecule or between molecules, so that it is free from cleavage by exonuclease I, or with RNA. It is considered that the annealing between the antisense DNA for detection and the promoter sequence part of the random DNA probe before the memory preparation was not inhibited among those that formed a partial complementary strand and remained uncut. In addition, it is considered that those that were not treated with exonuclease I were not inhibited from annealing with the promoter sequence of the random DNA probe before memory preparation by the antisense DNA for detection. By storing the memory in the random sequence portion, it is considered that the transcription initiation mechanism by the promoter operates efficiently and the transcription function of the present invention operates correctly.

  Further, FIG. 6 compares an unpurified memory DNA probe prepared from 100 pmol random DNA probe, a purified memory DNA probe, and a 50 pmol random DNA probe (control 2) without exonuclease cleavage. The memory DNA probe of 100 mer or more contained in the unpurified memory DNA probe is estimated to be about 0.5 pmol, but shows a fluorescence stronger than 50 pmol of the random DNA probe under the same conditions. However, 3 pmol memory DNA probe shows stronger fluorescence. From these, it is expected that by purifying a memory DNA probe having a length of 100 mer or more, the specificity increases and the detection sensitivity of the memory DNA probe can be increased. Furthermore, as described above, by comparing with the random DNA probe (control 2) not subjected to the exonuclease treatment of FIG. 5, in the random DNA probe, the promoter portion is not effective because of higher order structure formation between the probes and inside the probe. It is presumed that the amount of fluorescence detected is lowered because the polymerase cannot recognize. Regarding the high specificity of the memory DNA probe for the target RNA, fluorescence observation data of the purified memory DNA probe (g to l in FIG. 4) and the random DNA probe prepared as a control 1 before the memory preparation (a in FIG. 4). ~ F) will be compared. Since the purified memory DNA probe initially prepared in 2 volumes exceeded the measurable fluorescence of the plate reader, it was diluted 2 times and measured again. In the presence of RNA, by making a complementary strand with RNA where RNA is present, a promoter sequence important for detection reaction can be protected. It can be seen that the amount of fluorescence can be detected 7 times (FIG. 6). The fragile hybridization ability of random DNA probes cannot sufficiently prevent exonuclease cleavage and higher-order structure formation in the molecule that inhibits fluorescence observation, so it is estimated that strong fluorescence like memory DNA probes cannot be obtained. Is done. Combined with the fact that such sensitivity was not achieved in preliminary experiments using unpurified memory DNA probes, these results indicate that the method of the present invention is more sensitive to purification and stores the sequence information of the sample nucleic acid, It was found that the presence or absence of a target nucleic acid having stored sequence information can be determined by the amount of fluorescence.

  When the target nucleic acid is RNA transcribed from a cancer marker gene such as PPAP2B or PIM1, the presence or absence of disease can be diagnosed by detecting the target nucleic acid. The method of the present invention is an excellent system that can determine a plurality of similarity patterns possessed by a plurality of sample nucleic acids without determining the sequence in advance, and can be applied to library preparation.

  By the method of the present invention, sequence information of an arbitrary nucleic acid can be stored on a nucleic acid molecule even if the sequence is not specified. This is effective in removing the sequence information stored from a huge amount of data including noise as noise, creating a library, and efficiently classifying the presence pattern of the target nucleic acid. This makes it easy to exhaustively search for biomarkers that are effective for disease diagnosis, expands disease regions for which genetic diagnosis is effective, and enables expansion of disease regions for which early detection and early treatment are possible.

Moreover, the target nucleic acid in a sample can be detected simply and with high sensitivity using the sequence information and the fluorescence detection function stored as described above. Furthermore, based on the nucleic acid pattern classified by the method of the present invention, a memory nucleic acid probe library can be prepared to easily diagnose a subject or a population of subjects exhibiting the target nucleic acid pattern, which is effective in the field of diagnostic medicine. It is.

  In addition, the method of the present invention has the potential to be widely applied in medical situations, and is currently used to diagnose and treat psychiatric / neurological diseases or cancer and other systemic diseases for which specific gene expression is predicted to occur. And is expected to be effective in prevention.

An example of the configuration of a random nucleic acid probe and a memory nucleic acid probe and a procedure for producing a memory nucleic acid probe using the random nucleic acid probe are shown. The detection procedure of the target nucleic acid using a memory nucleic acid probe is shown. The detection mechanism using the fluorescence emission of the detection reporter molecule transcribed from the sequence encoding the detection reporter molecule will be described using a malachite green aptamer as an example. An outline of an example of preparing a library of memory nucleic acid probes is shown. The production of a microarray using a library of memory nucleic acid probes is shown. An outline of an example of diagnosis using a library of memory nucleic acid probes is shown. An outline of a target RNA detection experiment using a memory DNA probe using a random DNA probe using pTRI-β-Actin-Mouse RNA as a control is shown. The transcription reaction liquid obtained using the random DNA probe to which malachite green was added, the transcription reaction liquid obtained using the random DNA probe to which malachite green was not added, and the fluorescence intensity of malachite green alone are shown. Differences in RNA detection function between purified and unpurified memory DNA probes are shown by comparison with random DNA probes.

SEQ ID NO: 1, 2, 4, 6, 8-16 and 18: synthetic DNA
SEQ ID NOs: 3, 5, 7, and 17: synthetic RNA
N at positions 60 to 79 of SEQ ID NO: 8 represents an arbitrary base SEQ ID NO: 17 at position 100 represents a biotinylated base SEQ ID NO: 18 at position 100 represents 2-amino-6- (2- Thienyl) purine or 2-amino-6- (2-thiazolyl) purine

Claims (37)

The following steps:
(A) contacting a nucleic acid probe comprising a promoter sense sequence and a random sequence consisting of 5 to 100,000 bases with a sample nucleic acid to form a double strand between the random sequence of the nucleic acid probe and the sample nucleic acid;
(B) decomposing one strand of the nucleic acid probe from the 3 ′ end of the nucleic acid probe to a portion where the nucleic acid probe and the sample nucleic acid form a double strand;
(C) extending a sequence complementary to the sequence of the sample nucleic acid from the portion where the nucleic acid probe and the sample nucleic acid form a double strand to the 3 ′ end side of the nucleic acid probe using the sample nucleic acid as a template;
(D) removing the sample nucleic acid and recovering a nucleic acid probe containing a sequence complementary to the sequence of the sample nucleic acid;
A method for producing a nucleic acid probe having sequence information of a sample nucleic acid. The method according to claim 1, wherein the sample nucleic acid is a body fluid, a cell or a tissue, or a nucleic acid collected from them. The method according to claim 2, wherein the body fluid is saliva, blood or urine. The method according to any one of claims 1 to 3, wherein the nucleic acid is DNA or RNA. The method according to any one of claims 1 to 4, wherein the nucleic acid probe is DNA and the sample nucleic acid is RNA. The method according to any one of claims 1 to 5, wherein the promoter sequence is a T7 promoter sequence, a T3 promoter sequence or an SP6 promoter sequence, or a tandem sequence in which these sequences are linked. The method according to any one of claims 1 to 6, wherein the single-strand degradation in step (b) is performed by exonuclease I. The method according to any one of claims 1 to 7, wherein the extension of the sequence in step (c) is reverse transcription or replication. The method according to any one of claims 1 to 8, wherein the nucleic acid probe further comprises a sense sequence encoding a detection reporter molecule downstream of the promoter sense sequence. 10. The method of claim 9, wherein the detection reporter molecule is an aptamer RNA molecule. The method according to claim 10, wherein the reporter molecule for detection is a malachite green aptamer RNA molecule. The method according to any one of claims 1 to 11, wherein a plurality of nucleic acid probes having sequence information of each sample nucleic acid are prepared using a plurality of sample nucleic acids. The method according to any one of claims 1 to 12, wherein the sequence of the sample nucleic acid is not specified. The following steps:
(A) contacting a sample with a nucleic acid probe comprising a promoter sense sequence and a sequence complementary to all or part of the target nucleic acid, and forming a double strand between the nucleic acid probe and the target nucleic acid contained in the sample Forming step,
(B) a step of decomposing a single strand of the nucleic acid probe from the 3 ′ end of the nucleic acid probe, wherein when the nucleic acid probe and the target nucleic acid form a double strand, Decomposing the nucleic acid probe to a part forming a chain;
(C) a detection antisense nucleic acid comprising a sequence complementary to the promoter sense sequence and an antisense sequence encoding a detection reporter molecule is added, and a double strand is formed between the promoter sense sequence and the complementary sequence Forming steps,
(D) adding a polymerase to transcribe an antisense sequence encoding the detection reporter molecule to form a detection reporter molecule;
(E) confirming the transcription reaction in step (d) and detecting a target nucleic acid in the sample;
A method for detecting a target nucleic acid in a sample, comprising: The method according to claim 14, wherein the nucleic acid probe used in step (a) is prepared by the method according to any one of claims 1 to 13. The method according to claim 14 or 15, wherein the sample is a body fluid, a cell, a tissue, or a nucleic acid collected from them. The method according to claim 16, wherein the body fluid is saliva, blood or urine. The method according to any one of claims 14 to 17, wherein the nucleic acid is DNA or RNA. The method according to claim 18, wherein the nucleic acid probe is DNA and the target nucleic acid is RNA. The method according to any one of claims 14 to 19, wherein the promoter sequence is a T7 promoter sequence, a T3 promoter sequence or an SP6 promoter sequence, or a tandem sequence in which these sequences are linked. 21. The method according to any one of claims 14 to 20, wherein the polymerase of step (d) is T7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase. The method according to any one of claims 14 to 21, wherein the single-strand degradation in step (b) is performed by exonuclease I. The detection reporter molecule is a malachite green aptamer RNA molecule, and the target nucleic acid in the sample is detected using malachite green as a detection reagent for confirming the transcription reaction. Method. The nucleic acid probe further comprises a sense sequence encoding a detection reporter molecule downstream of the promoter sense sequence, and in step (c), the sense sequence encoding the detection reporter molecule and the detection reporter molecule in the detection antisense nucleic acid are encoded. The method according to any one of claims 14 to 23, wherein a double strand is formed between the antisense sequence and the antisense sequence. The method according to any one of claims 14 to 24, wherein the sequence of the target nucleic acid is not specified. Kit for detecting a target nucleic acid comprising the following (1) and (2):
(1) a nucleic acid probe comprising a promoter sense sequence and a random sequence comprising 5 to 100,000 bases, or a nucleic acid probe comprising a promoter sense sequence and a sequence complementary to all or part of the target nucleic acid ,
(2) A detection antisense nucleic acid comprising a sequence complementary to a promoter sense sequence and an antisense sequence encoding a detection reporter molecule . The kit according to claim 26, wherein the random sequence consists of 10 to 1000 bases. 28. The kit according to claim 26 or 27, wherein the nucleic acid probe is DNA or RNA. The kit according to any one of claims 26 to 28, wherein the promoter sequence is a T7 promoter sequence, a T3 promoter sequence or an SP6 promoter sequence, or a tandem sequence in which these sequences are linked. 30. The kit according to any one of claims 26 to 29, wherein the nucleic acid probe further comprises a sense sequence encoding a detection reporter molecule downstream of the promoter sense sequence. The kit according to any one of claims 26 to 30, wherein the antisense sequence encoding the detection reporter molecule is capable of forming a malachite green aptamer molecule by transcription thereof. 32. The method according to any one of claims 26 to 31, further comprising at least one selected from the group consisting of exonuclease I, enzyme for probe extension (reverse transcription or replication), RNA polymerase, substrate and detection reagent. Kit. The following steps:
(A) producing a memory nucleic acid probe having nucleic acid sequence information derived from a reference subject using the method according to any one of claims 1 to 13;
(B) bringing the memory nucleic acid probe into contact with a nucleic acid derived from a subject exhibiting a specific state or characteristic, and forming a double strand with a nucleic acid having the same sequence information as that of the memory nucleic acid probe;
(C) obtaining a nucleic acid group specific to a subject exhibiting a specific state or characteristic by removing the memory nucleic acid probe;
A method for producing a library of genes specific to a subject exhibiting a specific state or characteristic, comprising: Reference subject is healthy or normal subjects, claim 3 3 The method described. It is a specific condition disease or disorder, according to claim 3 3 or 3 4 The method according. A reference subject nucleic acid is mRNA from a subject showing a nucleic acid and a particular state or features derived from the method according to any one of claims 3 3-3 5. Further, (d) from the nucleic acid group specific to the subject exhibiting the specific condition or characteristic, to the subject exhibiting the specific condition or characteristic using the method according to any one of claims 1 to 13. 4. A memory nucleic acid probe library having gene sequence information specific to a subject exhibiting a specific state or characteristic is produced by performing a step of producing a memory nucleic acid probe having unique sequence information. the method according to any one of the 3-3 6.

Images