CN101526466B - Fluorescence biological sensing technology based on interaction between terminal-protected analyzed detected small molecules of exonuclease III and combined protein - Google Patents
Fluorescence biological sensing technology based on interaction between terminal-protected analyzed detected small molecules of exonuclease III and combined protein Download PDFInfo
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- CN101526466B CN101526466B CN2009100431302A CN200910043130A CN101526466B CN 101526466 B CN101526466 B CN 101526466B CN 2009100431302 A CN2009100431302 A CN 2009100431302A CN 200910043130 A CN200910043130 A CN 200910043130A CN 101526466 B CN101526466 B CN 101526466B
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- exonuclease iii
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- oligonucleotide dna
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Abstract
The invention discloses a fluorescence biological sensing technology based on the interaction between terminal-protected analyzed detected small molecules of exonuclease III and combined protein. The technology comprises the interaction between an oligonucleotide DNA chain modified by terminal organic small molecules and the protein, the protective analysis of the exonuclease III, and the fluorescence proportional detection of the oligonucleotide DNA chain in a double-chain structure based on double-chain indicating dye for terminal protection. The invention utilizes the small molecules modified at the terminal of the oligonucleotide DNA chain 3' to be combined with the specificity of the combined protein or antibody, is based on the terminal protective function of the exonuclease III, interacts the small molecules and the protein and detects the small molecules or the combined protein by the fluorescence of the protected oligonucleotide DNA chain and the double-chain embedded dye. The method has the advantages of simple and convenient operation, rapid economy and high sensitivity and specificity so as to be a general technology for the safety detection of food and agricultural products, environmental poison detection, drug screening, and the like.
Description
Technical field
The invention belongs to a kind of biosensor technique that detects organic molecule and binding protein interactions, comprise that the oligonucleotide DNA chain of terminal organic molecule modification and the interaction and the exonuclease III of albumen protect the fluorescent quantitation detection of analyzing and carrying out the duplex structure oligonucleotide DNA chain of terminal protection based on double-stranded indicating dye.
Background technology
Organic molecule such as organic molecule and binding protein interactions, medicine and chemical toxicant and the protein-bonded rapid screening of micromolecule are extremely important for fields such as clinical diagnosis, medical research, food and public safety, drug screening, environmental monitorings with detecting.The organic molecule commonly used at present and the detection technique of binding protein interactions mainly contain surface plasma resonance, the complementary segment immunoassay of enzyme and fluorescence anisotropy analysis etc.These detection techniques need complicated operations, accurate instrument, and cost is higher.
Summary of the invention
The technical problem to be solved in the present invention is; deficiency at the prior art existence; a kind of biological sensing technology based on exonuclease III terminal protection analyzing and testing micromolecule and binding protein interactions is proposed; it does not have dependence to dna sequence dna; can detect the combination of micromolecule and albumen; also can detect, or detect micromolecule by competitive reaction in conjunction with albumen, and easy and simple to handle, quick, sensitive.
Technical scheme of the present invention is that described biological sensing technology based on exonuclease III terminal protection analyzing and testing micromolecule and binding protein interactions comprises:
(1) interaction of the oligonucleotide DNA chain of terminal organic molecule modification and albumen and exonuclease III protection are analyzed;
(2) fluorescent quantitation that carries out the duplex structure oligonucleotide DNA chain of terminal protection based on double-stranded indicating dye detects.
Below the present invention made further specify.
The oligonucleotide DNA chain that described terminal organic molecule is modified and the interactional detection of albumen and exonuclease III protection are analyzed and are adopted following steps to realize:
Step for micromolecule protein-bonded interactional detection with it is: the storing solution of getting described small numerator modified duplex structure DNA oligonucleotide chain places micro tube; Add then and comprise the protein-bonded sample solution of micromolecule; Add exonuclease III reaction again;
For the detection of organic molecule, the step that adopts competitive reaction to detect is: the storing solution of getting described small numerator modified duplex structure DNA oligonucleotide chain places micro tube; Add then and comprise micromolecular sample solution to be measured, mix the back and add certain density micromolecular in conjunction with albumen or (mouse source) monoclonal anti liquid solution; Add exonuclease III reaction again.
The detection by quantitative of the duplex structure oligonucleotide DNA chain of described terminal protection can adopt the double-stranded fluorescence intercalative dye indicator method of standard to detect.
Among the present invention, the modification of the terminal organic molecule of described duplex structure oligonucleotide DNA realizes by existing cross-linking reaction technology.For example, for the organic molecule that has carboxyl, can adopt 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide and succinimide cross-linking reaction technology and 3 ' end NH
2The oligonucleotide DNA strand that comprises the protoheme aptamer of mark carries out crosslinked; For the amino organic molecule of band, the cross-linking reaction technology that can adopt 4-(N-maleimide ylmethyl) cyclohexane-1-carboxylic acid succinimide ester is carried out crosslinked with the oligonucleotide DNA strand that comprises the protoheme aptamer that is marked with sulfydryl with 3 ' end.The cross-linking reaction product can be used the dialysis purifying.
Further, among the present invention, the oligonucleotide DNA chain that described terminal organic molecule is modified and the interactional detection of albumen and exonuclease III protection are analyzed and are adopted following steps to realize:
A. the step for micromolecule protein-bonded interactional detection with it is: the storing solution of getting the described small numerator modified duplex structure DNA oligonucleotide chain of 5 μ l places micro tube, add 1.5 μ l, 10 * exonuclease III reaction buffer solution, this solution is 10mM Bis Propane-HCL, pH 7.0,10mM MgCL, 1mM DTT); Add 8 μ l then and comprise the protein-bonded sample solution of micromolecule, 37 ℃ of isothermal reactions 0.5 hour; Add 10U exonuclease III again, place 37 ℃ of reactions after 5 minutes, be warming up to 80 ℃ of reactions and carried out hot deactivation in 20 minutes to stop enzyme reaction;
B. for the detection of organic molecule, the step that adopts competitive reaction to detect is: the storing solution of getting the described small numerator modified duplex structure DNA oligonucleotide chain of 5 μ l places micro tube, add 1.5 μ l, 10 * exonuclease III reaction buffer solution, this solution is 10mM Bis Propane-HCL, pH 7.0,10mM MgCL, 1mM DTT); Add 5 μ l then and comprise micromolecular sample solution to be measured, mix the back and add certain density micromolecularly in conjunction with albumen or (mouse source) monoclonal anti liquid solution 3 μ l, the monoclonal antibody final concentration is 5 times of the small numerator modified oligonucleotide DNA chain concentration that adds; 37 ℃ of isothermal reactions 0.5 hour, add 10U exonuclease III again, place 37 ℃ of reactions after 5 minutes, be warming up to 80 ℃ of reactions and carried out hot deactivation in 20 minutes to stop enzyme reaction.
Notice that above reaction conditions is an optimal conditions, described liquor capacity all can change at double and not change optimal result.Change ratio or reagent addition sequence can make the protection efficient of organic molecule modified oligonucleotide dna single chain change in 1%～100%.
Among the present invention; the detection by quantitative of the duplex structure oligonucleotide DNA chain of described terminal protection can adopt the double-stranded fluorescence indicator method of standard to carry out, and below is with the detection by quantitative step of double-stranded fluorescence intercalative dye SYBR-GREEN 1 indicator method to the hair clip type oligonucleotide DNA chain of end protection:
In described protective reaction solution; add 5 μ l SYBR-GREEN 1 (a kind of double-stranded fluorescence intercalative dye, dilution in 1: 30000), be diluted to 100 μ l with the exonuclease reaction buffer; putting into fluorescence spectrophotometer, is that excitation wavelength, 520nm are that emission wavelength is surveyed its fluorescence letter intensity with 480nm.
The present invention proposes the method that the terminal protection of a kind of DNA oligonucleotides based on duplex structure is analyzed, the method does not have dependence to dna sequence dna, only need design a hairpin-shaped oligonucleotide probe or design a pair of complementary two strands, carry out the micromolecule mark, can realize detecting at its 3 ' end; It also can be directly used in the protein-bonded detection of micromolecule, also can be by the competition of oligonucleotide DNA chain and antibodies indirect detection organic molecule of micromolecule in the sample solution and micromolecule mark, its operation is fast and convenient, sensitive special, and being expected provides a current techique platform for clinical diagnosis, medicament research and development, medicine toxicological analysis, environmental monitoring etc.
Embodiment:
Embodiment 1: based on the terminal protection of exonuclease analyzing and testing Ochratoxin A
1) 3 ' end NH
2The oligonucleotide DNA chain of mark and Ochratoxin A crosslinked
The Ochratoxin A that takes by weighing 9.3mg is dissolved in 2.5mL phosphate buffered solution (0.1M NaH
2PO
4, pH 7.4), add 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC) of 1mg again, at room temperature stir 15min, add 2.8mg succinimide (NHS) again, room temperature reaction 30 minutes; The Ochratoxin A solution of getting after 260 μ l activate joins 3 ' the end NH that 260 μ l concentration are 1 μ M
2In the solution of the hair clip type oligonucleotide DNA chain of mark (sequence is 5 '-GAA TTC CAA GCGCGA AGT TTT CTT CGC GCT TGG AAT TC-3 '), transferring its pH with the NaOH of 1M is 7.4, and room temperature reaction is 2 hours under gentle agitation; The reaction back adds 3.6mg oxammonium hydrochloride cessation reaction in mixed liquor.
The cross-linking reaction product is moved on in the dialysis tubing of 1mL, the molecular cut off of dialysis tubing is 6000D.Dialysis tubing is put into 500ml phosphate buffered solution (0.1M NaH
2PO
4, pH 7.4) in 4 ℃ of dialysis 12 hours, changed a dislysate every 3 hours, dialyse with ultrapure water for the last time.Oligonucleotide DNA chain after the dialysis is in charge of in the refrigerator that is stored in-20 ℃ standby.The oligonucleotide DNA chain of-20 ℃ of above-mentioned preservations is with 10 times of aqua sterilisa dilutions and be stored in 4 ℃ of refrigerators standby as secondary storing solution.
2) detection of Ochratoxin A
The secondary storing solution of oligonucleotide DNA strand of getting above-mentioned Ochratoxin A mark is in micro tube, final concentration is 100nM, add 5 μ l SYBR-GREEN 1 (a kind of double-stranded fluorescence intercalative dye, dilution in 1: 30000) and 10 * exonuclease III of 1.5 μ l react buffer solution (10mM Bis Propane-HCL, pH 7.0,10mM MgCL
21mM DTT), add 5 μ l Ochratoxin A sample solution to be detected again, mixing back adding 3 μ l concentration is the Ochratoxin A mouse resource monoclonal antibody of 1000nM, room temperature reaction 15 minutes, the reaction back adds 8U exonuclease III (NEB company) after 37 ℃ of enzymes are cut 30 minutes, is warming up to 80 ℃ of reactions and carries out hot deactivation in 20 minutes to stop enzyme reaction; Cutting end-product to 100 μ l with exonuclease reaction buffer dilution enzyme, put into fluorescence spectrophotometer, is that excitation wavelength, 520nm are that emission wavelength is surveyed its fluorescence letter intensity with 480nm.
By above-mentioned 1), 2) step detects the Ochratoxin A standard solution (concentration is done 7 different concentration altogether from 1nM to 200nM) of 7 preparations, writes down the fluorescence intensity of each standard solution sample.With fluorescence intensity level Ochratoxin A concentration mapping in the standard solution is obtained typical curve.
Embodiment 2: based on exonuclease end points protection analyzing and testing FABP
1) 3 ' end NH
2The oligonucleotide DNA chain of mark and folic acid crosslinked
The folic acid that takes by weighing 10mg is dissolved in 2.5mL phosphate buffered solution (0.1M NaH
2PO4, pH 7.4), add 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide (EDC) of 1mg again, at room temperature stirred 15 minutes, add 2.8mg succinimide (NHS) again, room temperature reaction 30 minutes; The folic acid solution of getting after 260 μ l activate joins 3 ' the end NH that 260 μ l concentration are 1 μ M
2(sequence: in the solution 5 '-GAATTC CAA GCG CGA AGTTTT CTT CGC GCT TGG AAT TC-3 '), transfer its pH with the NaOH of 1M is 7.4 to the hair clip type oligonucleotide DNA strand of mark, and room temperature reaction is 2 hours under gentle agitation; The reaction back adds 3.6mg oxammonium hydrochloride cessation reaction in mixed liquor.
The cross-linking reaction product is moved on in the dialysis tubing of 1mL, the molecular cut off of dialysis tubing is 6000D.Dialysis tubing is put into 500ml phosphate buffered solution (0.1M NaH
2PO4, pH 7.4) in 4 ℃ of dialysis 12 hours, changed a dislysate every 3 hours, dialyse with ultrapure water for the last time.The oligonucleotide DNA chain that oligonucleotide DNA chain after the dialysis is in charge of standby above-mentioned-20 ℃ of preservations in the refrigerator that is stored in-20 ℃ is with 10 times of aqua sterilisa dilutions and be stored in 4 ℃ of refrigerators standby as secondary storing solution.More than all reactions all under the condition of shading, carry out.
2) detection of FABP:
The secondary storing solution of oligonucleotide DNA chain of getting the above-mentioned folic acid mark of 5 μ l is in micro tube, the concentration that makes final DNA chain is 100nM, add 5 μ l SYBR-GREEB 1 (a kind of double-stranded fluorescence intercalative dye, dilution in 1: 30000) and 10 * exonuclease III of 1.5 μ l react buffer solution (10mM Bis Propane-HCL, pH 7.0,10mM MgCL
21mMDTT), add 5 μ l FABP sample solution to be detected again, room temperature reaction 15min, the reaction back adds 8U exonuclease III (NEB company product) after 37 ℃ of enzymes are cut 30min, is warming up to 80 ℃ of reaction 20min and carries out hot deactivation to stop enzyme reaction.Cutting end-product to 100 μ l with exonuclease reaction buffer dilution enzyme, put into fluorescence spectrophotometer, is that excitation wavelength, 520nm are that emission wavelength is surveyed its fluorescence letter intensity with 480nm.
By above-mentioned 1), 2) step detects the FABP standard solution (concentration is from 1nM to 200nM) of 7 preparations, the fluorescence intensity that writes down each standard solution sample is done the typical curve of the concentration and the fluorescence intensity of FABP, and its concentration is determined in fluorescence intensity that FABP produced in the unknown sample and the contrast of the curve of standard.
Claims (1)
1. biological method for sensing based on exonuclease III terminal protection analyzing and testing micromolecule and binding protein interactions is characterized in that this method comprises:
(1) interaction of the oligonucleotide DNA chain of terminal organic molecule modification and albumen and exonuclease III protection oligonucleotide DNA chain are analyzed the oligonucleotide DNA chain of protection then;
Step for micromolecule protein-bonded interactional detection with it is: the storing solution of getting described organic molecule modification duplex structure DNA oligonucleotide chain places micro tube; Add then and comprise organic micromolecular protein-bonded sample solution; Add exonuclease III reaction again;
For the detection of organic molecule, the step that adopts competitive reaction to detect is: the storing solution of getting described organic molecule modification duplex structure DNA oligonucleotide chain places micro tube; Add then and comprise micromolecular sample solution to be measured, mix the back and add certain density micromolecular in conjunction with albumen or monoclonal anti liquid solution; Add exonuclease III reaction again;
(2) based on double-stranded indicating dye duplex structure oligonucleotide DNA chain is carried out end protection, the fluorescent quantitation of the duplex structure oligonucleotide DNA chain protected is detected; Described detection adopts the double-stranded fluorescence intercalative dye indicator method of standard to carry out.
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