JP3996097B2 - Fluorescence sensor using RNA-peptide complex - Google Patents

Description

【図面の簡単な説明】
【図１】本発明の実施の一形態であるＡＴＰ検出用リボヌクレオペプチドセンサーの作製法を概念的に説明する図である。
【図２】上記の作製法により実際に得られた、ＡＴＰとの結合能の高いＲＮＡ配列群のうち、相同性のある１グループの配列を示す図である。
【図３】上記ＲＮＡ配列群のうち、相同性のある他のグループの配列を示す図である。
【図４】上記ＲＮＡ配列群のうち、相同性のあるさらに他のグループの配列を示す図である。
【図５】（ａ）〜（ｆ）は、複数種類のＡＴＰ検出用リボヌクレオペプチドセンサーについて、ＡＴＰ添加に伴う蛍光変化を評価した結果を示すグラフである。
【図６】（ａ）〜（ｄ）は、１種類のＡＴＰ検出用リボヌクレオペプチドセンサーについて、各種ＮＴＰ添加に伴う蛍光変化を評価した結果を示すグラフである。
【図７】１種類のＡＴＰ検出用リボヌクレオペプチドセンサーについて、AMP, ATP, dATP添加に伴う相対蛍光強度変化を評価した結果を示すグラフである。
【図８】ＲＮＡサブユニットが互いに異なる２種類のＡＴＰ検出用リボヌクレオペプチドセンサーについて、ＡＴＰ添加に伴う蛍光変化割合を評価した結果を示すグラフである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a fluorescence sensor using an RNA-peptide complex. The present invention enables high-throughput detection of various molecules such as biomolecules such as second messengers and neurotransmitters, environmental pollutants and endocrine disrupting substances (environmental hormones) present in the environment, and substances in food and drink. As a new molecular detection technology, it is expected to be used in a wide range of industrial fields such as analysis of in vivo dynamics of various biomolecules, various clinical tests and diagnoses in the medical field, environmental research (environmental measurement / environment monitoring), food analysis it can.
[0002]
[Prior art]
In post-genomic science, innovative discoveries can be expected by analyzing the in vivo dynamics of various molecules such as molecules responsible for intracellular information transmission and associating the results with genomic information. However, the analysis methods for that purpose have been very limited, and for example, in order to elucidate the entire signal transduction, there is an urgent need to develop molecular sensors or biosensors that can observe the dynamics of second messengers, etc. in the cell. It was.
[0003]
So far, research has been actively conducted to synthesize artificial receptors that capture second messengers using organic molecules in Japan and overseas. However, few have reached satisfactory levels in terms of both selectivity and affinity, and methods for producing molecular sensors and biosensors based on artificial receptors have not been established.
[0004]
Regarding the method of producing an artificial receptor, the present inventor previously described a ribonucleopeptide live designed based on the three-dimensional structure of a complex of a human immunodeficiency virus (HIV) Rev peptide and RRE (Rev Response Element) RNA. It has been reported that a tailor-made receptor can be constructed from a rally using an in vitro selection method (see Patent Document 1 and Non-Patent Document 1 below).
[0005]
[Patent Document 1]
JP 2003-33186 A
[Non-Patent Document 1]
Morii, T., Hagihara, M., Sato, S., and Makino, K. (2002) J. Am. Chem. Soc. 124, 4617-4622
[0006]
[Problems to be solved by the invention]
The above receptor production method developed by the present inventor is a versatile method combining a structure-based design method and a combinatorial method. Therefore, there are many highly selective ribonucleopeptides for second messengers and neurotransmitters. It is possible to make a receptor. The present inventor further considered that this technology could be applied and developed to convert the artificial receptor selected by the above-described receptor production method into a biosensor. If this is realized, it will be possible to create highly selective molecular sensors of tailor-made for various molecules, so the field of application is not limited to the use in research and analysis of signal transduction mechanisms, but also in the medical field, environmental field, Expected to be used in a wide range of industries such as food sanitation.
[0007]
The present invention has been made in view of the above problems, and its purpose is to develop a new molecular sensor using an RNA-peptide complex (ribonucleopeptide) and to various molecules using the sensor. To provide a detection method and an apparatus system.
[0008]
[Means for Solving the Problems]
In view of the above problems, the present inventor has intensively studied whether it is possible to construct a biosensor in which fluorescence intensity changes upon binding to a substrate molecule by introducing a fluorescent molecule into an RNA-peptide complex, By modifying the peptide subunit of the RNA-peptide complex that binds highly selectively to a biologically active molecule, adenosine triphosphate (ATP), with a fluorescent molecule, ATP whose fluorescence intensity changes greatly upon ATP binding Succeeded in producing a fluorescent biosensor. In addition, it has been found that by using such a sensor, a substrate molecule such as ATP can be visualized and quantified by fluorescence in real time, and the present invention has been completed.
[0009]
That is, the present invention includes the following inventions A) to L) as medically and industrially useful methods and products.
A) A ribonucleopeptide fluorescent sensor obtained by labeling (or modifying) a complex of an RNA subunit and a peptide subunit with a fluorescent molecule.
B) The ribonucleopeptide fluorescent sensor according to A) above, wherein the substrate binding region of the complex or the vicinity thereof is labeled with a fluorescent molecule.
C) The ribonucleopeptide fluorescent sensor according to A) or B) above, wherein the N-terminal amino group of the peptide subunit is labeled with a fluorescent molecule.
D) The ribonucleo described in any of A) to C) above, wherein the fluorescent molecule is selected from the group consisting of pyrene, NBD fluoride, coumarin and dansyl chloride. Peptide fluorescence sensor.
E) The RNA subunit is the following base sequence: ggucugggcgca- (N) n-ugacgguacaggcc (in the base sequence (N) n indicates a substrate binding region, “N” is arbitrarily selected from adenine, uracil, guanine and cytosine The ribonucleopeptide fluorescent sensor according to any one of A) to D) above, wherein “n” is an integer of 20 to 30).
F) The ribonucleopeptide fluorescent sensor according to any one of A) to E) above, wherein the peptide subunit has an amino acid sequence derived from the Rev protein of human immunodeficiency virus (HIV).
G) The ribonucleopeptide fluorescent sensor according to any one of A) to F) above, wherein the substrate is adenosine triphosphate (ATP).
H) The ribonucleopeptide fluorescent sensor according to E) above, wherein the substrate is adenosine triphosphate (ATP), and (N) n consists of a base sequence represented by SEQ ID NO: 1 or 2.
I) A method for detecting a substrate molecule by using the ribonucleopeptide fluorescent sensor according to any one of A) to H) above by changing the fluorescence intensity.
J) The method according to I) above, which is used for detecting a biomolecule or a molecule in the environment or food and drink.
K) A molecular sensor element used in the method described in I) or J) above and comprising one or a plurality of ribonucleopeptide fluorescent sensors and a carrier thereof.
L) An apparatus that is used in the method described in I) or J) and includes one or a plurality of ribonucleopeptide fluorescence sensors and detection means for detecting changes in the fluorescence intensity.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, specific embodiments and technical scope of the present invention will be described in detail.
[0011]
(1) Ribonucleopeptide fluorescent sensor of the present invention
FIG. 1 is a diagram conceptually illustrating a method for producing a ribonucleopeptide sensor for ATP detection according to an embodiment of the present invention. As shown in the figure, in this production method, a peptide subunit of an ATP-binding ribonucleopeptide receptor (hereinafter sometimes referred to as “RNP receptor”) is labeled with a fluorescent molecule, and a ribonucleopeptide sensor ( Hereinafter, it may be referred to as “RNP sensor”). Since the RNP receptor is composed of an RNA subunit and a peptide subunit, for example, by replacing one of the peptide subunits with a fluorescent peptide, the function of the RNP receptor can be modified to an RNP sensor relatively easily. it can.
[0012]
The RNP sensor needs to change its fluorescence intensity by binding to a substrate molecule. As shown in FIG. 1, the RNP sensor of the present embodiment has an N-terminal amino group (—NH) of a peptide subunit that is expected to be located in the vicinity of the substrate binding region. ₂ ) Was labeled with a fluorescent molecule having an amine reactive group. As a result, an RNP sensor in which a change (increase) in fluorescence intensity, which is considered to be caused by a change in the surrounding environment of the fluorescent molecule with the binding of ATP, was successfully observed (details will be described in Examples described later). To do).
[0013]
Thus, the fluorescent molecule is preferably introduced into the N-terminal amino group of the peptide subunit or the side chain of the amino acid residue on the N-terminal side, but is not limited thereto. For example, the RNA subunit may be modified with a fluorescent molecule instead of the peptide subunit. In addition, the fluorescent molecule is preferably introduced at or near the substrate binding region. However, as long as the change in the fluorescence intensity of the RNP sensor is measured as a result of binding to the substrate, the fluorescent molecule is separated from the substrate binding region. It may be introduced into the site.
[0014]
A fluorescent molecule is a molecule that is excited by an appropriate light energy (excitation wavelength) and emits light, and its luminescence change is relatively easy to measure by the apparatus, and reacts with a peptide subunit or an RNA subunit. There is no particular limitation as long as it can be directly bonded to this or indirectly bonded via another substance. In the examples described later, three types of fluorescent molecules, namely pyrene (pyrene: 1-pyrenesulfonyl chloride) and NBD fluoride (NBD fluoride: 4-fluoro-7-nitrobenz-2-oxa-1). , 3-Diazole (4-fluoro-7-nitrobenz-2-oxa-1,3-diazole), and 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride: 5-dimethylaminonaphthalene-1-sulfonyl chloride) chloride)) to produce an RTP sensor for ATP detection. Of course, other fluorescent molecules may be used. The change in fluorescence due to the binding of the substrate is considered to vary depending not only on the introduction position of the fluorescent molecule but also on the type of the fluorescent molecule, so an appropriate one may be selected from various fluorescent molecules.
[0015]
As an example of a method for optimizing the introduction position of the fluorescent molecule and the type of the fluorescent molecule, the following method can be mentioned. That is, using multiple types of fluorescent molecules, creating a peptide library with different modification positions of each fluorescent molecule to the peptide subunit, and combining it with the RNA subunit of the receptor, A labeled RNP sensor is made. From these, an optical sensor that responds most efficiently to each substrate may be selected.
[0016]
As with the RNP receptor, the RNP sensor of the present invention can design and produce an RNP sensor using any molecule as a substrate, as with the RNP receptor. Basically, it is the same as the method of manufacturing the RNP receptor described in JP-A-2003-33186. That is, an RNA subunit in which a peptide binding region and a substrate binding region having a random base sequence are bound is prepared, a complex of the RNA subunit and the peptide subunit is formed, and the complex is obtained by an in vitro selection method. And a target substrate are reacted, and then a complex having a binding ability to the substrate is selected. Next, RNA is recovered from the selected complex, and the recovered RNA is converted into DNA by a reverse transcription reaction. After this is amplified by PCR, a new RNA subunit is prepared. Based on the RNA subunit, Again, an RNA-peptide complex having a higher affinity for the substrate is formed by repeating the operation of forming a complex with the peptide subunit and selecting a complex having the binding ability to the substrate by an in vitro selection method. Is elected.
[0017]
In other words, a ribonucleopeptide molecule having a binding ability to a target substrate, such as ATP, is selected from among a library of ribonucleopeptides that are diversified by a random RNA base sequence portion using, for example, ATP. A new ribonucleopeptide library is prepared by selecting and amplifying them. In this way, by repeating the selection from many types of molecular species, an RNA-peptide complex having a high binding ability capable of binding more selectively to the target substrate can be produced.
[0018]
Preferred examples of the peptide subunit include those having an amino acid sequence derived from the Rev protein of HIV, and preferred examples of the peptide binding region of the RNA subunit include those having a Rev Response Element (RRE). it can. In particular, the RNA subunit has the following base sequence: ggucugggcgca- (N) n-ugacgguacaggcc As a suitable example, an RNA containing a single type of base, wherein “n” is an integer of 20 to 30) can be mentioned. The peptide subunit has the following 17 amino acid sequence: TR (X) ₂ RRN (X) ₃ R (X) ₆ (Preferably, a peptide subunit having the following amino acid sequence: TRQARRNRRRRWRERRQR is preferred.
[0019]
2 to 4 show the nucleotide sequence of the RNA subunit substrate binding region in the RNA-peptide complex actually obtained by the selection method using the above-mentioned in vitro selection method and having high binding ability to ATP. It is. The obtained RNA sequences were classified into three types from the viewpoint of homology. Moreover, in the method implemented this time, since the random base sequence part was set to 30 bases, the length of the RNA sequence obtained as a substrate binding region is also about 30 bases.
[0020]
In Examples described later, by introducing a fluorescent molecule into a complex of an RNA subunit (430RNA / 428RNA) having the sequences of 430 and 428 in FIG. 4 as a substrate binding region and a peptide subunit (Rev peptide), respectively. An RTP sensor for ATP detection was obtained. The 430RNA has the following sequence (a) as a base sequence including a substrate binding region, and the 428RNA has the following sequence (b) as a base sequence including a substrate binding region. The peptide subunit has the amino acid sequence of (c) below.
(A) ggucugggcgca-uauaccguagugguuuguguguggguugg-ugacgguacaggcc (SEQ ID NO: 3)
(B) ggucugggcgca-agggcuuggugugccgauuucggggcauuu-ugacgguacaggcc (SEQ ID NO: 4)
(C) TRQARRNNRRRWRERRQ (SEQ ID NO: 5)
[0021]
In Examples described later, an RNA subunit (430RNA / 428RNA) having high binding ability to ATP is finally obtained by an in vitro selection method, and then this 430RNA or 428RNA and a peptide subunit (Rev peptide) are converted into fluorescent molecules. A complex with the fluorescently modified peptide modified in step 1 is formed to produce an RTP sensor for ATP detection. As other methods, for example, peptide subunits or RNA subunits are labeled with fluorescent molecules in advance, and in vitro selection is performed using the peptide subunits or RNA subunits thus fluorescently labeled. An RNP sensor may be made.
[0022]
(2) Usefulness of the present invention (application field)
As described above, the RNP sensor of the present invention can be designed and manufactured in a tailor-made manner as a sensor for an arbitrary molecule, and is excellent in versatility. For example, it can be used as follows.
[0023]
(A) A second messenger cell under various physiological conditions by designing and producing the RNP sensor of the present invention so as to specifically bind to a specific second messenger and introducing it into the cell by microinjection or the like. Internal dynamics can be optically detected and measured in real time for quantification. As described above, the RNP sensor of the present invention is useful for analyzing in vivo kinetics as a sensor for small organic molecules in a living body.
[0024]
(B) The RNP sensor of the present invention can be used as a molecular sensor for various biomolecules such as physiologically active substances such as ATP, second messengers, and neurotransmitters, but its use is not limited to detection of biomolecules. In addition, it can be used for various substance detections such as detection of environmental pollutants and endocrine disrupting substances (environmental hormones) present in the environment, and detection of specific substances in food and drink. As described above, the industrial field in which the present invention can be used is extremely wide. For example, it can be expected to be used for various clinical examinations and diagnoses in the medical field, environmental research (environmental measurement / environmental monitoring), food analysis, and the like. .
[0025]
(C) In addition, by applying microarray fabrication technology, etc., the RNP sensors of the present invention are arranged in high density in an array, and a molecular sensor element (such as a molecular sensor chip) that simultaneously detects many molecules at high throughput is fabricated. Is possible. Such a molecular sensor element is also included in the present invention. The molecular sensor element of the present invention basically comprises one or a plurality of RNP sensors and a carrier thereof (such as a substrate for fixing or holding the RNP sensor), thereby detecting one or a plurality of substances. I just need it. When detecting a plurality of substances, each RNP sensor may be labeled with different fluorescent molecules according to the target substance. In addition, the RNP sensor of the present invention may be used for detecting a substance having a relatively large molecular weight (such as an oligopeptide), and the molecular sensor element of the present invention is promising among next-generation chip technologies. It can also be applied to “protein chips”.
Of course, the molecular sensor element of the present invention can be used in various fields described in (B) above, and can be used in various applications such as a clinical diagnostic chip and an environmental contamination detection chip.
[0026]
(D) A new intermolecular interaction analysis apparatus or substance detection apparatus using the RNP sensor of the present invention or the molecular sensor element of the present invention can be realized, and such an apparatus is also included in the present invention. The apparatus basically has only to be configured to include one or a plurality of RNP sensors and detection means for detecting changes in the fluorescence intensity. For example, (1) means for introducing, adding or mixing the RNP sensor of the present invention (or the molecular sensor element of the present invention) into a sample such as a biological sample, and then the RNP sensor (or the book) taken out from the sample or from the sample (2) means for guiding the sample to the surface of the RNP sensor (or the molecular sensor element of the present invention), which is fixed to the RNP sensor (or the molecular sensor element of the present invention). Then, the structure provided with the means to measure the fluorescence of a RNP sensor (or molecular sensor element), etc. are mentioned.
[0027]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited at all by these Examples.
[0028]
First, materials and methods used in each example described later will be described.
〔reagent〕
Side chain-protected Fmoc (9-fluorenylmethoxycaebonyl) amino acids were purchased from Novabiochem, Fmoc-PAL-PEG resin, HATU from Applied Biosystems, and DMF for peptide synthesis from Wako Pure Chemical. Fluorescence introduction reagents (5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride), 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD fluoride), 1-pyrenesulfonyl chloride) are purchased from Molecular Probe did. ATP agarose resin and nucleotides (ATP, ADP, AMP, dATP, UTP, CTP, GTP, 8-bromo-ATP) were purchased from Sigma-Aldrich. Klenow DNA Polymerase, restriction enzymes (BamHI, EcoRI) and T4 RNA Ligase were purchased from New England Biolab. Pyrobest DNA Polymerase and TaKaRa Ligation Kit Ver.2 were purchased from TaKaRa. DH5α competent cells for library creation were purchased from Invitrogen. Reverse transcriptase (AMV (Avian Myeloblastosis Virus) Reverse Transcriptase) was purchased from Promega. An RNA transcription kit (AmpliScribe T7 High Yield Transcription Kit) was purchased from Epicentre. BactoTrypton, Yeast Extract, and BactoAgar were purchased from Difco. The synthesis of DNA library and DNA for PCR primers was requested from Amersham Pharmacia. QIAprep Spin Miniprep Kit was purchased from Qiagen. Other reagents used were for molecular biology and of special grade.
For PCR, BIO-RAD iCycler Thermal Cycler or TaKaRa PCR Thermal Cycler PERSONAL is used. For standard PCR reaction, TaKaRa Pyrobest DNA Polymerase is used, and 0.2 mM primer DNA in the attached Pyrobest Buffer II reaction solution is used. Using 0.2 mM dNTP conditions.
[0029]
[Preparation of DNA library]
A template DNA (RREN30) (5'-GGAATAGGTCTGGGCGCACA-N30-TGACGGTACAGGCCGAAAG-3 ') containing a random base sequence of 30 bases with primer REV01 (5'-CTTTCGGCCTGTACCGTCA-3') complementary to the 3 'end region of the template DNA After annealing at 2 ° C. for 2 hours, double-stranded DNA was synthesized by Klenow DNA Polymerase at 25 ° C. for 15 minutes. Subsequently, using a primer (5'-TCTAATACGACTCACTATAGGAATAGGTCTGGGCGCA-3 ') containing a T7 promoter and a primer complementary to the 3' end region of the template DNA, denaturation with Pyrobest DNA Polymerase at 94 ° C for 30 seconds, annealing at 55 ° C A DNA library containing 30 strands of random double strands was constructed by performing a PCR reaction for 30 seconds at 72 ° C for 1 minute at 72 ° C.
[0030]
[Preparation of RNA library]
Using 1 mg of double-stranded DNA as a template, RNA was transcribed in a 20 ml volume at 37 ° C. for 3 hours (according to the Epicentre Ampli Scribe T7 Transcription Kits protocol). After completion of the transcription reaction, 1 U DNase was added and reacted at 37 ° C. for 15 minutes to decompose the template DNA. The transcribed RNA was ethanol precipitated in the presence of sodium acetate. The RNA sample was dissolved in an 80% formamide aqueous solution, heat-treated at 80 ° C. for 5 minutes, denatured by rapid cooling on ice, and purified on an 8% polyacrylamide denaturing gel containing 6M urea. RNA detected by UV on a TLC plate was cut out and extracted with TE buffer (10 mM Tris-HCl (pH 7.6), 1 mM EDTA). The extracted RNA was ethanol precipitated in the presence of sodium acetate. The purified RNA is diluted to a buffer containing 10 mM Tris-HCl (pH 7.6) and 150 mM NaCl to a concentration of 10 mM, heated at 80 ° C. for 3 minutes, and then slowly cooled to form an appropriate secondary structure over 2 hours to room temperature. I tried to do it.
[0031]
(Rev peptide synthesis)
Chemical synthesis was performed by Fmoc solid phase synthesis. As the resin, Fmoc-PAL-PEG resin (0.41 mmol / g) was used. Condensation was performed by adding 5 equivalents of Fmoc amino acid and 5 equivalents of HATU dissolved in DMF containing 5% diisopropylethylamine to the resin and stirring for 30 to 60 minutes. After removing the Fmoc group of the N-terminal amino acid, the N-terminal of the peptide was acetylated using N-methylimidazole and acetic anhydride. Cutting out from the resin and deprotection of the side chain was performed by adding phenol (0.75g), thioanisole (0.5ml), ethanedithiol (0.25ml), water (0.5ml), TFA (10ml) on ice under nitrogen atmosphere. The mixed solution was added to the dried resin and stirred for 6 hours on ice in a nitrogen atmosphere. After completion of the reaction, water was added on ice and the mixture was stirred for 30 minutes, extracted and purified with ether, gel filtration (Sephadex G-10) was performed using 5% aqueous acetic acid as an eluent, and purified by reverse phase HPLC.
[0032]
[Method for selecting ATP-binding ribonucleopeptide]
ATP resin (2.0 mmolATP / ml resin capacity) fixed with 4% beaded agarose was used for the binding experiment. The resin is pre-binding buffer (10 mM Tris-HCl (pH 7.6), 100 mM NaCl, 10 mM MgCl ₂ ) Was equilibrated by washing 3 times with a binding buffer of 3 times the resin volume. To a 50 ml resin, 1 mM RNA and 1.5 mM Rev peptide were added in a 100 ml volume and allowed to bind on ice for 30 minutes with occasional stirring. After the reaction, the resin was washed 3 times with 300 ml of binding buffer. The RNA bound to the resin was eluted with 100 ml of buffer containing 4mMATP, and this was repeated three times. RNA was collected by ethanol precipitation in the presence of ammonium acetate. RNA was dissolved in 10 ml TE (10 mM Tris-HCl (pH 7.6), 1 mM EDTA), and 1.5 ml of RNA was used as a template, Promega kit (Reverse Transcription System The reverse transcription reaction was performed at 42 ° C. for 30 minutes in the presence of the REV01 primer. The reaction solution was heat treated at 99 ° C. for 5 minutes to inactivate reverse transcriptase, and then a portion of the reaction solution was denatured using the REV01 primer and FOR01 primer (5′-GAATTCTAATACGACTCACTATAGG-3 ′). PCR was performed at 30 ° C. for 30 seconds, annealing at 55 ° C. for 30 seconds, and extension reaction at 72 ° C. for 1 minute. The reaction solution was collected every 5 cycles, and the amplified DNA was analyzed by 8% polyacrylamide electrophoresis. PCR was performed with the minimum number of cycles in which DNA amplification was confirmed exponentially by analysis with polyacrylamide electrophoresis, and a new DNA library was prepared. The PCR product was purified by 2-propanol precipitation in the presence of ammonium acetate and subjected to the next selection.
[0033]
[Synthesis of Rev peptide with fluorescent molecule introduced at N-terminus]
Rev peptide was synthesized by Fmoc solid-phase synthesis method as described above, and the resin with the N-terminal protected with Fmoc group was immobilized on DMF containing 30% piperidine for 5 minutes x 3 times. The Fmoc group was removed. The resin was thoroughly washed with DMF, and then reacted for 3 hours at room temperature with 10 equivalents of fluorescent molecules dissolved in DMF containing 5% DIEA. Unreacted fluorescent molecules were thoroughly washed away with DMF, washed with methanol and ether, and then thoroughly dried. The dried resin was reacted with a mixed solution of phenol (0.75 g), thioanisole (0.5 ml), ethanedithiol (0.25 ml), water (0.5 ml), and TFA (10 ml) for 6 hours at room temperature. At the same time of cutting, deprotection was performed. After completion of the reaction, water was added on ice and the mixture was extracted and purified with ether, and then gel filtration (Sephadex G-10) was performed using 5% aqueous acetic acid as an eluent, followed by purification by reverse phase HPLC. Peptides were identified by MALDI-TOF mass spectrum. The peptide concentration was determined by amino acid composition analysis.
Dansyl-Rev calculated for [M +] 2478.8, found 2480.5;
Pyrene-Rev calculated for [M +] 2593.0, found 2594.6; NBD-Rev peptide was decomposed by laser irradiation and could not be identified by mass spectrum, but it was confirmed to be a single peak in HPLC.
[0034]
[RNA sequence analysis of ATP-binding ribonucleopeptide]
For the DNA library selected 10 times, in order to add a restriction enzyme cleavage region, the FOR02 primer (5'-CGgaattcTAATACGACTCACTATAGG-3 ', lowercase notation is EcoRI recognition sequence), REV02 primer ( PCR was performed using 5′-GCGggatccTTTCGGCCTGTACCGTCA-3 ′ (lowercase notation is BamHI recognition sequence). PCR was performed in 100 ml reaction volume using 100 ng of template DNA for 4 cycles under conditions of denaturation at 94 ° C. for 30 seconds, annealing at 55 ° C. for 30 seconds, and extension reaction at 72 ° C. for 1 minute. The PCR product was purified by 2-propanol precipitation in the presence of ammonium acetate, and then subjected to an enzyme reaction with BamHI and EcoRI at 37 ° C. for 3 hours. After enzymatic digestion, it was treated with phenol and chloroform and purified by ethanol precipitation in the presence of sodium acetate. A 3-fold molar amount of PCR product was added to the pUC19 plasmid DNA previously cleaved by BamHI and EcoRI reactions, and a ligation reaction was performed at 16 ° C. for 1 hour using TaKaRa Ligation Kit ver.2. The ligation sample was directly added to DH5 competent cells for transformation, and cultured overnight on LB ampicillin medium. The transformed E. coli was cultured in 5 ml of liquid LB medium containing ampicillin. The culture solution cultured for 16 hours was centrifuged at 3,000 rpm for 10 minutes to recover E. coli. From the recovered E. coli, plasmid DNA was recovered by the alkaline method according to the protocol of Qiagen MiniPrepKit.
Prepare the recovered plasmid DNA (200 ng), M13RV primer (5'-CAGGAAACAGCTATGAC-3 ', 1.6 pmol), 4 ml of BigDyeTerminator premix solution to a reaction volume of 10 ml, denature at 96 ° C for 30 seconds, and anneal at 50 The reaction was performed for 25 cycles at 15 ° C. for 15 seconds and at 60 ° C. for 4 minutes. After completion of the reaction, it was purified by ethanol precipitation in the presence of sodium acetate solution. The sample was dissolved in a sequencing loading solution, treated at 99 ° C. for 2 minutes, then rapidly cooled on ice, and half of the amount was used for analysis. DNA sequence analysis was performed according to the manufacturer's protocol using an automated DNA sequence analyzer (ABI Model 377) from Applied Biosystem.
[0035]
[Preparation of RNA from sequence-analyzed plasmid DNA]
Using 10 ng of the plasmid DNA for which the sequence was confirmed, denaturation was performed at 94 ° C for 30 seconds using FOR03 primer (5'-GAATTCTAATACGACTCACTATAGG-3 ') and REV04 primer (5'-GGAATAGGCCTGTACCGTC-3') for annealing for 30 seconds. The reaction was carried out for 20 cycles at 55 ° C. for 30 seconds and the extension reaction at 72 ° C. for 1 minute. The PCR product was subjected to 2-propanol precipitation in the presence of ammonium acetate. The purified DNA was dissolved in TE, RNA was transferred by the method described above, and then purified using a denaturing gel.
[0036]
[Measurement using ATP fluorescence sensor]
The fluorescence spectrum was measured using a Hitachi spectrofluorometer F-4500. During reaction conditions (10 mM Tris-HCl (pH 7.6), 100 mM NaCl, 10 mM MgCl ₂ ) After the complex of the purified RNA and the fluorescence-modified peptide 0.5 to 1 mM was formed, various nucleotides were added and incubated at room temperature for 30 minutes to measure fluorescence. NBD-Rev peptide was measured at an excitation wavelength of 470 nm, Dansyl-Rev peptide was measured at 340 nm, and Pyrene-Rev peptide was measured at an excitation wavelength of 360 nm. The slit widths of the excitation wavelength and the fluorescence wavelength were each 5 nm and measured at 20 ° C.
[0037]
[Example 1: Construction of ATP-binding ribonucleopeptide receptor]
First, a ribonucleopeptide receptor was prepared for adenosine triphosphate (ATP). As described above, a ribonucleopeptide pool obtained by mixing 1: 1 with a Rev peptide was prepared using a random sequence containing 30 bases as described above. The RNA sequence of the substrate binding region in the ribonucleopeptide receptor obtained using the ATP resin immobilized with 2 ′ and 3 ′ ribose by the in vitro selection method is shown in FIGS.
[0038]
[Example 2: Construction of RNA subunit of ATP-binding ribonucleopeptide receptor]
For the RNA subunits (428RNA and 430RNA) each having the 428 and 430 sequences in Fig. 4, they form complexes with NBD-, Dansyl-, and Pyrene-Rev peptides, respectively, and perform ATP titration. I investigated. RNA was prepared using primers designed so that 5 ′ and 3 ′ completely formed complementary strands. The base sequences of the substrate binding regions of 428RNA and 430RNA are as follows.
430 UAUACCGUAGUGGUUUGUGUGUGGGUUGG (SEQ ID NO: 1)
428 AGGGCUUGGUGUGCCGAUUUCGGGGCAUUU (SEQ ID NO: 2)
[0039]
[Example 3: Evaluation of ATP detection performance of RNP sensor prepared using Pyrene peptide, NBD peptide or Dansyl peptide and 428RNA or 430RNA]
During reaction conditions (10 mM Tris-HCl (pH 7.6), 100 mM NaCl, 10 mM MgCl ₂ ) After forming a 1: 1 complex of RNA (428RNA or 430RNA) and fluorescent modified peptide (Pyrene-Rev peptide 0.5μM, NBD-Rev peptide 0.5μM, Dansyl-Rev peptide 1.0μM), add ATP Fluorescence was measured after incubation at room temperature for 30 minutes. NBD-Rev peptide was measured at an excitation wavelength of 470 nm, Dansyl-Rev peptide was measured at 340 nm, and Pyrene-Rev peptide was measured at an excitation wavelength of 360 nm. In this way, 428RNA or 430RNA was used to form complexes with various peptides to produce RNP sensors, and changes in fluorescence associated with the addition of ATP were evaluated. The result is shown in FIG. In the figure, (a) to (c) are prepared using 428 RNA, (d) to (f) are each prepared using 430 RNA, (a) and (d) are pyrene, (b) and (e) are NBD, (C) and (f) are the fluorescence accompanying the ATP addition (0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000, 3000, 10000 μM) of RNA sensors prepared using dansyl as the fluorescent molecule. The result of intensity change is shown. In each graph, the vertical axis represents fluorescence intensity, and the horizontal axis represents wavelength (nm).
As shown in the figure, the change was small in the RNP sensor using the Dansyl peptide, but in the RNP sensor prepared using the NBD peptide and Pyrene peptide, the RNP sensor prepared using either 428 RNA or 430 RNA. Even in the case, the fluorescence intensity increased with the addition of ATP.
[0040]
[Example 4: Performance evaluation of RNP sensor (base selectivity evaluation)]
In order to evaluate the recognition of the base moiety, ATP, UTP, CTP, and GTP were evaluated for fluorescence changes in the concentration range evaluated by ATP. During reaction conditions (10 mM Tris-HCl (pH 7.6), 100 mM NaCl, 10 mM MgCl ₂ ) After forming a 1: 1 complex of RNA (428RNA) and fluorescent modified peptide (Pyrene-Rev peptide 0.5μM), various NTPs were added (0.1, 0.3, 1, 3, 10, 30, 100, 300, And the fluorescence was measured at an excitation wavelength of 360 nm. The result is shown in FIG. As shown in the figure, when UTP, CTP, or GTP was added, no fluorescence change was observed, indicating that the RNP sensor retained high base recognition ability. In each graph of FIG. 6, the vertical axis indicates the fluorescence intensity, and the horizontal axis indicates the wavelength (nm).
[0041]
[Example 5: Selectivity of RNP sensor (comparison of dATP, AMP, ATP)]
In order to confirm the selectivity, the fluorescence change accompanying the addition of ligand (AMP, ATP, or dATP) was measured for the complex of 428 RNA and Pyrene-Rev peptide, and the relative fluorescence intensity (ie, fluorescence intensity after addition of ligand ( I) is the fluorescence intensity (I ₀ Plotted against the ligand concentration plus. The result is shown in FIG. As shown in the figure, when AMP and dATP were added, changes in fluorescence intensity and affinity were similar to ATP, indicating that the base part of adenosine was accurately recognized.
[0042]
[Example 6: Comparison of RNP sensor performance using different RNA subunits]
The ATP binding of the ribonucleopeptide sensor prepared using 428RNA or 430RNA as the RNA subunit and Pyrene-Rev peptide as the peptide subunit was analyzed from the change in fluorescence when ATP was added to the ribonucleopeptide sensor. The result is shown in FIG. Measurement conditions (10 mM Tris-HCl (pH 7.6), 100 mM NaCl, 10 mM MgCl ₂ The dissociation constant (Kd) of the ATP and ribonucleopeptide sensor at) was 39 μM for 428 RNA and 86 μM for 430 RNA. In the graph of FIG. 8, black circles indicate the measurement results for the 428RNA-Pyrene-Rev complex, and black triangles indicate the measurement results for the 430RNA-Pyrene-Rev complex.
[0043]
【The invention's effect】
As described above, the present invention relates to a fluorescence sensor using an RNA-peptide complex, and as described above, it can be designed and produced in a tailor-made manner as a sensor for any molecule, and is excellent in versatility. Expected to be used in a wide range of industrial fields such as medical and environmental fields.
[Sequence Listing]

[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram conceptually illustrating a method for producing a ribonucleopeptide sensor for ATP detection according to an embodiment of the present invention.
FIG. 2 is a diagram showing one group of homologous sequences among a group of RNA sequences with high binding ability to ATP actually obtained by the above-described production method.
FIG. 3 is a diagram showing sequences of other groups having homology among the RNA sequence groups.
FIG. 4 is a view showing sequences of still another group having homology among the RNA sequence groups.
FIGS. 5A to 5F are graphs showing the results of evaluating fluorescence changes associated with the addition of ATP for a plurality of types of ribonucleopeptide sensors for ATP detection.
FIGS. 6A to 6D are graphs showing the results of evaluating fluorescence changes associated with various NTP additions for one type of ATP-detecting ribonucleopeptide sensor.
FIG. 7 is a graph showing the results of evaluating changes in relative fluorescence intensity associated with addition of AMP, ATP, and dATP for one type of ribonucleopeptide sensor for ATP detection.
FIG. 8 is a graph showing the results of evaluating the rate of change in fluorescence associated with the addition of ATP for two types of ATP detection ribonucleopeptide sensors having different RNA subunits.

Claims (7)

A ribonucleopeptide fluorescent sensor formed by labeling a complex of an RNA subunit and a peptide subunit with a fluorescent molecule,
The RNA subunit following nucleotide sequence: ggucugggcgca- (N) (N) n in n-ugacgguacaggcc (nucleotide sequence represents the substrate binding region, are selected, "N" is adenine, uracil, and any of guanine and cytosine One type of base, " n " is an integer from 20 to 30),
The peptide subunit has an amino acid sequence derived from the Rev protein of human immunodeficiency virus (HIV);
The fluorescent molecules are selected from pyrene (pyrene) or NBD fluoride (NBD fluoride), and ribonucleoprotein peptide fluorescence sensor, characterized in that coupled to the N-terminal amino group of the peptide subunits.   The ribonucleopeptide fluorescent sensor according to claim 1, wherein the substrate is adenosine triphosphate (ATP).   The ribonucleopeptide fluorescent sensor according to claim 2, wherein the substrate is adenosine triphosphate (ATP), and (N) n consists of a base sequence shown in SEQ ID NO: 1 or 2.   A method for detecting a substrate molecule by using the ribonucleopeptide fluorescent sensor according to any one of claims 1 to 3 based on a change in fluorescence intensity thereof.   The method according to claim 4, wherein the method is used for detecting a biomolecule, or a molecule in the environment or food and drink.   A molecular sensor element used in the method according to claim 4 or 5, comprising one or a plurality of ribonucleopeptide fluorescent sensors and a carrier thereof.   6. An apparatus comprising one or more ribonucleopeptide fluorescent sensors and a detection means for detecting a change in the fluorescence intensity used in the method according to claim 4 or 5.

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