JP4788942B2 - Deoxyribozymes for specific species detection - Google Patents

Description

  The present invention relates to a method for detecting, identifying or quantifying RNA unique to a biological species using deoxyribozyme, and the same reagent.

In recent years, hammerhead ribozymes, hairpin ribozymes, deoxyribozymes (DNA enzymes) and nucleic acid molecules having a catalytic function have been studied. Among them, deoxyribozyme is being studied as an interesting new technique in biotechnology because of its simple structure, high stability, and low cost.
By artificially designing deoxyribozyme and cleaving RNA specifically in the base sequence, cell gene expression can be freely controlled, and it is expected to overcome many genetic diseases and viral diseases. In fact, there have been many reports on the application of deoxyribozymes to medicines (see Patent Documents 1 to 3).

  In the previous studies, the application of deoxyribozymes has been mostly directed at cleaving messenger RNA in living cells from the viewpoint of controlling gene expression. Recently, a method for analyzing the secondary structure of a target RNA using the catalytic activity of deoxyribozyme immobilized on a sensor chip has been reported (see Patent Document 4). However, to date, there has been no report on the detection, identification, and quantification of biological species present in a sample by using an RNA sequence unique to the biological species as a target nucleic acid.

In addition, a method for identifying and quantifying biological species in a sample using a biomolecular interaction sensor chip provided with an oligonucleotide probe that hybridizes with a 16S rRNA sequence specific to the biological species has been developed (non-patented). Reference 1).
However, since this method detects a signal based on the presence or absence of hybridization, it has a problem that not only a lot of false positives are detected but also a lack of quantitativeness.

Special Table 2002-525037 Special Table 2002-534117 Special table 2003-506078 JP2003-265168 Nelson, B. P., Liles, M. R., Frederick, K. B., Corn, R. M., and Goodman R. M. (2002) ”Environ. Microbiol.” 4, 735-743

  An object of the present invention is to provide a means for easily, rapidly and accurately detecting, identifying or quantifying RNA in a sample containing at least one species-specific RNA.

  As a result of diligent research to solve the above-mentioned problems, the present inventors have used a deoxyribozyme that recognizes and cleaves the base sequence portion of RNA unique to a biological species as a reagent, acts on an RNA-containing sample, By measuring the presence and amount of RNA cleaved in the sample by ribozyme, it is possible to easily, quickly and accurately detect, identify, and quantify RNA specific to a biological species. The present invention has been found to be extremely effective in the detection and identification of specific species, and the present invention has been completed.

That is, the present invention is as shown in the following (1) to (16).
(1) A deoxyribozyme that recognizes and cleaves an RNA base sequence unique to a biological species is brought into contact with a sample containing an RNA fragment derived from at least one biological species, and whether or not the RNA fragment is cleaved. A method for detecting, identifying or quantifying RNA inherent in a biological species, characterized in that
(2) The method according to (1) above, wherein the ratio of the cut step piece to the uncut piece is calculated, and RNA unique to the biological species in the sample is quantified.
(3) The method according to (1) or (2) above, wherein a plurality of deoxyribozymes that recognize and cleave RNA base sequences unique to a biological species are used simultaneously.
(4) The method according to any one of (1) to (3) above, wherein the RNA fragment in the sample is amplified.
(5) The detection method according to (4) above, wherein the RNA fragment amplification method is any one of the NASBA method, TMA method, TRC method, and 3SR method.
(6) The method according to any one of (1) to (5) above, wherein the deoxyribozyme is immobilized on a solid phase alone.
(7) The method according to (6) above, wherein the solid phase carrier is a sensor chip for biomolecular interaction measurement.
(8) The method according to (7) above, wherein the biopolymer interaction measurement sensor chip is a surface plasmon resonance (SPR) sensor chip.
(9) The method according to any one of (1) to (8) above, wherein one or more nucleotides of deoxyribozyme are 2′-O-methylated.
(10) The method according to any one of (1) to (8) above, wherein one or more nucleotides of deoxyribozyme contains PNA (peptide nucleic acid).
(11) The method according to any one of (1) to (8) above, wherein one or more nucleotides of deoxyribozyme contains LNA (locked nucleic acid).
(12) The method according to any one of (1) to (11) above, comprising comparing the electrophoresis pattern of the RNA fragment-containing sample on which deoxyribozyme is allowed to act with the sample on which deoxyribozyme is not allowed to act .
(13) A method for detecting, identifying or quantifying a biological species comprising the method according to any one of (1) to (12) above.
(14) A reagent for detecting, identifying or quantifying RNA unique to a biological species, comprising a deoxyribozyme that recognizes and cleaves a base sequence unique to the biological species in RNA in a sample.
(15) The reagent according to (13) above, wherein a plurality of types of deoxyribozymes having different base sequences to be recognized are contained.
(16) A reagent for detecting, identifying or quantifying a specific species, characterized by comprising the deoxyribozyme according to (14) or (15) above.

  According to the present invention, since no labeling with a radioisotope or a fluorescent dye is required, a simpler and quicker analysis is possible. Further, since no special equipment is required for measuring them, it is economically advantageous.

In the present invention, deoxyribozyme means DNA having sequence-specific cleavage activity for single-stranded RNA, but its size is not particularly limited.
Examples of deoxyribozymes that can be particularly suitably used in the present invention include 8-17 DNA enzyme and 10-23 DNA enzyme shown in FIG. 1 (Santro, SW, and Joyce, GF (1997) Proc. Natl. Acad. Sci. USA 94, 4262-4266). These deoxyribozymes have a catalytically active site in the loop part, hybridize to the target RNA with the base sequence (recognition site) before and after that, and are shown in FIG. 1 of the target RNA in the presence of a divalent cation. It can cut at the position of the marked arrow.

The size of the deoxyribozyme used in the present invention is not particularly limited as long as it has catalytic activity, but the recognition site is preferably 10 to 30 bases in order to improve the specificity of substrate RNA sequence recognition. . The target RNA is RNA corresponding to a substrate when deoxyribozyme is viewed as an enzyme.
In the present invention, the deoxyribozyme to be used is designed based on the base sequence portion unique to the biological species in the RNA of the biological species to be analyzed. The base sequence of the target RNA need not be all known. In addition, even if it is not clear what kind of biological species the RNA in the sample to be analyzed is derived from, the deoxyribozyme designed as described above is allowed to act on the sample to cause the RNA to be analyzed in the sample. Can be confirmed from the presence or absence of the cleaved fragment, the presence of the biological species possessing it can be identified, and the identification and quantification thereof can also be performed.

  That is, according to the method of the present invention, various RNAs may be mixed in the sample regardless of whether or not the RNA in the sample is purified. For example, samples intended for measurement in complex microbial systems (mixed microbial RNA or genetic DNA, nucleic acids in soil, genes, etc.), or serum, urine, various biological cells containing RNA such as bacteria and viruses, Even if a culture solution of biological tissue is used as a sample, the presence of the target RNA to be analyzed can be accurately detected, identified and quantified by analyzing the RNA fragment cleaved based on the action of the deoxyribozyme used. This makes it possible to analyze the species.

In the present invention, in the case of detecting only RNA specific to a single species among RNAs specific to each species present in a sample, the base sequence portion inherent to the species to be analyzed is usually used. One type of deoxyribozyme designed based on the above may be used, but in order to further increase the accuracy, a deoxyribozyme that cleaves a plurality of sites with different base sequences can be used for one type of target RNA.
In the present invention, when a plurality of types of target RNA are present, one or more types of deoxyribozymes can be used simultaneously, thereby improving the analysis efficiency.
For example, when detecting a group of species similar to each other, a deoxyribozyme designed to cleave a sequence that is commonly present in the RNA of the group of species may be used.

The deoxyribozyme used in the present invention is composed of oligodeoxyribonucleotides, but these may be chemically modified. Specifically, deoxyribose having a hydrogen at the 2 ′ position substituted with an O-methyl group (2′-O-methylation) is used. Alternatively, LNA (locked nucleic acid) in which the entire deoxyribose is substituted may be used.
The deoxyribozyme used in the present invention may be PNA (peptide nucleic acid) having a skeleton in which the deoxyribose-phosphate skeleton, which is the skeleton structure of the nucleic acid skeleton, is substituted with glycine.

  The aforementioned LNA can be synthesized by a known method (Wengel, J. (1998) Acc. Chem. Res. 32, 301-310). 2'-O-methylation can be ordered from most DNA contract synthesis companies. In addition, PNA is readily available because Greiner Japan conducts custom synthesis. By applying such chemical modification to the oligonucleotide, the affinity with the target nucleic acid is increased, the hybridization between the deoxyribozyme and the target RNA is stabilized, and the cleavage efficiency is increased.

  Analysis of RNA-containing samples and biological species in the present invention can be performed, for example, as follows. RNA is extracted from a specimen containing at least one species collected from a certain environment to prepare a sample containing at least one species of RNA. Subsequently, deoxyribozyme designed based on the unique nucleotide sequence of RNA in the species to be analyzed is allowed to act on the sample containing RNA. In this case, preferably, the deoxyribozyme is allowed to act in a solution in which an RNA-containing sample is dissolved in a buffer solution usually used biochemically at an appropriate concentration, more preferably in the solution to which magnesium ions are added. good.

  After the reaction, RNA in the sample is separated by gel electrophoresis, and the position on the gel is confirmed by a nucleic acid staining reagent. Compare the electrophoresis pattern of the sample when deoxyribozyme is allowed to act with the electrophoresis pattern of the sample when deoxyribozyme is not acted. It can be determined that the specimen of the biological species does not include the biological species to be analyzed. In addition, if a band that is not in the former electrophoresis pattern is detected in the latter electrophoresis pattern, it can be understood that the specimen contains the species to be analyzed, and the detection and identification of the species in the specimen is performed. Can be done. Furthermore, if the RNA base sequence of the band is examined, it can be confirmed that it is a cleavage product by the deoxyribozyme that has been acted on. In this case, if the concentration of the band of the latter battle pattern is lower or disappears than the former electric pattern, it can be understood that the RNA corresponding to the band is cleaved.

In the present invention, the apparatus for analyzing RNA fragments may be any apparatus as long as it can measure the size and concentration of the band, and examples thereof include Gel Doc (BIO-RAD). Further, 2100 bioanalyzer (Agilent Technologies) can be cited as a particularly suitable apparatus for performing separation and analysis quickly.
In the present invention, by using these devices, in the case where a plurality of types of target RNAs are present, a single type of deoxyribozyme is allowed to act, and then the ratio of the uncut pieces to the cut pieces is calculated. It is possible to calculate the proportion of RNA fragments having an arbitrary sequence designed with deoxyribozyme in the total RNA fragments. Since this ratio reflects the ratio of the species to be analyzed in the specimen, the species to be analyzed can also be quantified.

The analysis method and analysis reagent of the present invention can be used in various fields such as medicine, molecular biology, and agriculture. In addition, it is said to be a complex microbial system or a symbiotic microbial system, and it is suitable for use with microorganisms that contain various microorganisms or that cannot be isolated from each other because at least one type of microorganism is mixed with cells from other animals or plants. it can.
In the synthesis of the deoxyribozyme of the present invention, the base sequence is designed based on the 5S rRNA, 16S rRNA, or 23S rRNA of a specific strain in a complex microorganism system or a symbiotic microorganism system, and the spacer region sequence of those rRNA sequences. be able to. By using this deoxyribozyme, the detection, identification and abundance of a specific strain in the system can be measured.

The abundance of an arbitrary RNA sequence can also be analyzed by measuring a change in signal generated based on cleavage of the target RNA of deoxyribozyme. Examples of means for measuring the signal change include a surface plasmon resonance (SPR) method and a crystal oscillator microbalance (QCM) method, but are not limited to these means.
In order to easily detect and quantify RNA fragments, deoxyribozyme may be immobilized on a sensor chip for measuring biopolymer interactions. In this case, labeling of the target RNA is generally unnecessary.

As a sensor chip used in the surface plasmon resonance (SPR) method, a normal surface plasmon resonance apparatus substrate can be used, and a commercially available one can be used.
In the present invention, the method for immobilizing deoxyribozyme on a solid support is not particularly limited, and any immobilization means that can be used for the production of an immobilization catalyst may be used.
The method of contacting the target RNA with deoxyribozyme immobilized on the sensor chip by this method is not particularly limited. Each step of the present method can be carried out using a commercially available surface plasmon resonance apparatus such as BIAcore3000 (Pharmacia Biosensor).
Examples of the present invention are shown below, but the present invention is not limited to these examples.

[Example 1]
Using total RNA derived from Escherichia coli K-12 MG1655 strain (hereinafter referred to as MG1655 strain) and Pseudomonas putida KT2440 strain (hereinafter referred to as KT2440 strain) as target RNA sequences, deoxyribozyme was allowed to act and cleaved specifically.

(1) A deoxyribozyme that specifically cleaves 16S rRNA of KT2440 strain was designed. Table 1 shows the sequence. The sequence indicated by the bold line is the catalytically active site. The synthesis and purification of oligodeoxyribonucleotides were performed by external contract. In addition, the cleavage site | part in the target RNA of the deoxyribozyme which has each base sequence of Table 1 was shown in FIG.

(2) MG1655 strain and KT2440 strain were cultured in LB medium, and total RNA containing 16S rRNA was extracted from the culture solution using Fast RNA Pro Blue Kit (Qbiogene), and this was used as a sample.
(3) Target sequence cleavage reaction with deoxyribozyme is performed at 37 ° C. under conditions of 500 ng target RNA and 5 μM deoxyribozyme in a buffer containing 50 mM Tris-Cl (pH 8.0) and 20 mM MgCl _2. Time reaction was performed. The cleavage reaction was terminated by mixing 100 mM EDTA and 8M urea.

(4) Target RNA and cleavage products were separated by electrophoresis on a 5% polyacrylamide / 8M urea denaturing gel. The reaction system for each lane is shown in Table 2 below.
A polyacrylamide gel stained with a shadow device Gel Doc (BIO-RAD) was photographed.
The results are shown in FIG. A cut piece was observed at the expected position, indicating that the target RNA can be cleaved at the target position. When total RNA derived from MG1655 strain was used as the target RNA, no cut pieces were observed.

Example 2
Quantitative analysis of the catalytic activity of deoxyribozyme was performed using MG1655 and KT2440-derived 16S rRNA fragments as target RNA sequences.
(1) MG1655 and KT2440 strains were cultured on LB agar medium, and one platinum loop was suspended in sterile distilled water.

The underlined part in Table 3 is the T7 promoter sequence. KT-FT7-F is a forward primer specific for 16S rDNA derived from KT2440 strain, and MG-FT7-F is a forward primer specific for 16S rDNA derived from MG1655 strain. FT7-R is a reverse primer specific for 16S rDNA from KT2440 and MG1655 strains.

(2) Using the 16S rRNA gene (16S rDNA) contained in the cell suspension as a template, 16S rDNA fragments derived from each strain were amplified by PCR. PCR was performed under the following conditions. The equipment used was icycler (BIO-RAD). Table 3 shows the primer sequences used.

・ Denaturation 95 ℃, 1 min
・ Annealing 55 ℃, 1 min
・ Elongation 72 ℃, 1 min
・ Taq polymerase: Taq Ampli Gold (Roche Applied Biotech.), 5 U
・ Primer addition concentration: 1 μM
・ DNTP concentration: 200 nM
・ Template nucleic acid: 1 μl of cell suspension
(3) The amplification product was purified using QIA quick PCR purification Kit (QIAGEN).
(4) T7 RiboMax Express Large Scale RNA Production System (Promega) was used for in vitro transcription at 37 ° C for 30 minutes to obtain 16S rRNA fragment. The reaction was performed under the following conditions.
・ Template DNA: 1 μg
・ Ribomax T7 Express buffer: 10 μl
・ Ribomax T7 Enzyme Mix: 2 μl
(5) The transcript was purified using the Purification with RNeasy MinElute Cleanup Kit and used as the target RNA.
(6) 16S rRNA of KT2440 strain and MG1655 strain were mixed at a ratio of Table 4 so that the total amount was 1000 ng, and deoxyribozyme cleavage reaction was performed.

(6) Target sequence cleavage reaction with deoxyribozyme was carried out at 37 ° C under conditions of 1000 ng of target RNA and 10 μM deoxyribozyme (KT629) in a buffer containing 50 mM Tris-Cl (pH 8.0) and 20 mM MgCl _2. The reaction was carried out for 1 hour. 100 mM EDTA was mixed to terminate the cleavage reaction.

(7) Target RNA and cleavage products were separated and quantified using 2100 bioanalyzer (Agilent Technologies).
Fig. 4 shows the electrophoresis results of the reaction system in which the target RNA KT2440 is 0%. Only the peak S of the substrate 16S rRNA was observed. FIG. 5 shows the electrophoresis results of the reaction system in which KT2440 was mixed at a ratio of 75%. In addition to the peak S of the substrate 16S rRNA, fragments P1 and P2 were observed.

The deoxyribozyme cleavage efficiency was calculated using the following formula.
Cutting efficiency (%) = (P1 + P2) / (P1 + P2 + S)
(Here, P1, P2, S indicate the area of each peak)
The amount of the cut pieces was quantified based on the electrophoresis results of the reaction performed at each mixing ratio. The abscissa indicates the abundance ratio of 16S rRNA derived from KT2440 strain among the target RNAs mixed at the start of the experiment, and the abscissa indicates the abundance ratio of 16S rRNA derived from KT2440 strain calculated from the analysis results after measurement. Was plotted on a graph representing. As shown in FIG. 6, a good correlation was obtained.

Example 3
Target RNA sequences include Escherichia coli (hereinafter referred to as E. coli), Pseudomonas aeruginosa (hereinafter referred to as P. aeruginosa), Enterobacter cloacae (hereinafter referred to as E. cloacae), Klebsiella pneumoniae (hereinafter referred to as K. pneumoniae), Enterococcus faecalis (as the target RNA sequence) E. faecalis), Staphylococcus aureus (hereinafter S. aureus), Staphylococcus epidermidis (hereinafter S. epidermidis), Staphylococcus haemolyticus (hereinafter S. haemolyticus), Staphylococcus hominis (hereinafter S. hominis) Cleavage experiments using deoxyribozymes specific to each species were performed according to the procedures (1) to (4).

(1) Deoxyribozymes that specifically cleave 16S rRNA of various strains were designed. Table 5 shows the sequence. The sequence indicated by the bold line is the catalytically active site.
Eco, Pae, Ecl, Kpn, Efa, Sau, Sep, Sha, and Sho are E. coli, P. aeruginosa, E. cloacae, K. pneumoniae, E. faecalis, S. aureus, S. epidermidis, S. haemolyticus, respectively. , A deoxyribozyme that specifically cleaves S. hominis. The synthesis and purification of these oligonucleotides were performed by an external contract.

(2) Each strain was cultured in NB medium, and total RNA containing 16S rRNA was extracted from the culture solution using Fast RNA Pro Blue Kit (Qbiogene), and this was used as a sample.
(3) Target sequence cleavage reaction with deoxyribozyme is carried out at 37 ° C under the conditions of 1 ug total RNA and 10 µM deoxyribozyme in a buffer containing 50 mM Tris-Cl (pH 8.0) and 20 mM MgCl ₂ . The reaction was performed for 1 hour. 100 mM EDTA was mixed to terminate the cleavage reaction.
(4) Target RNA and cleavage products were separated using 2100 bioanalyzer (Agilent Technologies). Table 6 shows the results. As a result of the action of the specific deoxyribozyme on each biological species, a fragment specific to the biological species was obtained. Further, as is apparent from these results, the means of the present invention can identify the pathogenic bacteria contained in the sample.

Example 4
Using total RNA from multiple species as the target RNA, multiple types of deoxyribozymes were allowed to act, and the cut fragments were analyzed. Table 7 shows the combinations of target RNA and deoxyribozyme used.
The analysis procedure is as shown in the following (1) to (3).

(1) Each strain was cultured in NB medium, and total RNA containing 16S rRNA was extracted from the culture solution using Fast RNA Pro Blue Kit (Qbiogene), and this was used as a sample.
(2) Target sequence cleavage reaction with deoxyribozyme was carried out at 37 ° C under the conditions of a total of 1 ug total RNA and 10 μM deoxyribozyme in a buffer containing 50 mM Tris-Cl (pH 8.0) and 20 mM MgCl _2. The reaction was performed for 1 hour. 100 mM EDTA was mixed to terminate the cleavage reaction.
(3) Target RNA and cleavage products were separated using 2100 bioanalyzer (Agilent Technologies).
The results are shown in FIGS. By using multiple species-specific deoxyribozymes at the same time, multiple species-specific peaks were detected in a sample containing multiple types of target RNA.

The analysis method of the present invention can be used in various fields such as medicine, molecular biology, and agriculture. It is said to be a complex microorganism system or a symbiotic microorganism system, and can be suitably used for microorganisms in which various microorganisms coexist or at least one type of microorganism is mixed with other animal and plant cells and cannot be isolated from each other. It can also be used for rapid detection of pathogenic bacteria in the field of clinical microorganisms.

The structure of a complex of deoxyribozyme and its target RNA is shown. Deoxyribozyme binds to its target RNA via Watson-Crick base pair at the substrate recognition site and cleaves the site indicated by the arrow. It is a figure which shows the cutting | disconnection site | part of each deoxyribozyme in 16S rRNA fragment | piece derived from Pseudomonasputida KT2440 strain. The results of electrophoresis using a 5% polyacrylamide denaturing gel after the RNA sequence-specific cleavage reaction with deoxyribozyme are shown in the photograph. It is a graph which shows the electrophoresis result by 2100Bioanalyzer after performing RNA sequence-specific cleavage reaction with deoxyribozyme. S shows the peak of the target RNA 16S rRNA. It is a graph which shows the electrophoresis result by 2100Bioanalyzer after performing RNA sequence-specific cleavage reaction with deoxyribozyme. S is the peak of the target RNA 16S rRNA, and P1 and P2 are the peaks of the cut pieces. The results of quantitative analysis of the target RNA by measuring the fragments obtained by deoxyribozyme are shown. It is a graph which shows the electrophoresis result by 2100Bioanalyzer after performing RNA sequence specific cleavage reaction with deoxyribozyme. As target RNA, mixed RNA derived from P. aeruginosa and S. aureus and deoxyribozyme mixed with Pse and Sau were used. It is a graph which shows the electrophoresis result by 2100Bioanalyzer after performing RNA sequence-specific cleavage reaction with deoxyribozyme. As target RNA, mixed RNA derived from P. aeruginosa and E. coli and deoxyribozyme mixed with Pse and Eco were used.

Claims (5)

  Contacting 5SrRNA, 16SrRNA, 23SrRNA or a deoxyribozyme that recognizes and cleaves the nucleotide sequence unique to the species in the spacer region of the rRNA sequence with a sample containing an RNA fragment derived from at least one species, Measuring the presence or absence of the RNA fragment and its amount, calculating the ratio of the cut piece to the uncut piece, and quantifying the RNA specific to the species in the sample, Species-specific RNA detection, identification and quantification methods.   2. The method according to claim 1, wherein 5SrRNA, 16SrRNA, 23SrRNA or a plurality of deoxyribozymes that recognize and cleave RNA base sequences unique to a biological species in a spacer region of these rRNA sequences are used simultaneously.   The method according to claim 1 or 2, comprising comparing the electrophoresis patterns of the RNA fragment-containing sample on which deoxyribozyme is allowed to act and the sample on which deoxyribozyme is not allowed to act.   A reagent for use in the method according to any one of claims 1 to 3, comprising 5SrRNA, 16SrRNA, 23SrRNA or a deoxyribozyme that recognizes and cleaves a base sequence unique to a biological species in a spacer region of these rRNA sequences. A reagent for detecting, identifying and quantifying RNA specific to a biological species.   The reagent according to claim 4, wherein a plurality of types of deoxyribozymes having different base sequences to be recognized are contained.