CN102533771B - Anti-tumor angiogenesis aptamer molecule and preparation method thereof - Google Patents
Anti-tumor angiogenesis aptamer molecule and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an anti-tumor angiogenesis aptamer molecule and a preparation method thereof. The anti-tumor angiogenesis aptamer molecule is characterized in that the sequence of the anti-tumor angiogenesis aptamer molecule is 5'-ACTTCCAGTCTATTCAATTGGGCCCGTCCGTATGGTGGGTGTGCGTGGCCACGAGTTTTAAAAAAAAAAACCAAGCTTTTTTTGATCATGT-3'. Cyclization modification design and preparation method processes which are performed aiming at linear deoxyribonucleic acid (DNA) aptamer molecules are built, and a method for efficiently and rapidly preparing annular DNA aptamers at a low cost is built through modified annular DNA aptamer molecules generated by RAC reaction.
Description
Technical field
The present invention relates to a kind of aptamer molecule and preparation method of Antineoplastic angiogenesis.
Background technology
Fit DNA or the RNA fragment being function and being equivalent to antibody, can carry out specific recognition and combination to target molecule, and effectively suppresses the biologic activity of target molecule.Fit nucleic acid sequence information obtains by exponential enrichment system evolution technology (SELEX) screening of part, and aptamer molecule can adopt chemical process low cost synthesis in enormous quantities.DNA aptamer molecule is highly stable in vitro, and lyophilized powder state normal temperature can preserve more than 1 year, therefore has good drug development prospect.Fitly at present be considered to one of nucleic acid drug having DEVELOPMENT PROSPECT most, other three kinds is RNAi, ribozyme and microRNA.Namely effectively, other three kinds of nucleic acid drugs that therefore relatively must play a role in cell, fit application prospect is more extensive for aptamer molecule and extracellular target molecule effect.Underway for the fit research of the Clinics and Practices aspect of the diseases such as hematologic disease, cardiovascular disorder, nervous system disorders, disease in the blood system, tumour at present, some are fit, and preparation enters clinical trial, the anti-Vascular Endothelial being specifically designed to treatment macular degeneration generates the factor (vascular endothelial growth factor, VEGF) APTAMER MEDICINAL (Macugen) and goes on the market.
In APTAMER MEDICINAL body, the feature of serum stability difference seriously hinders its clinical application.Owing to there is nuclease in blood, particularly exonuclease, made not add the linear DNA of any modification or the transformation period of RNA molecule less than 10 minutes, did not fitly also play desirable drug action and was namely degraded.Therefore will develop APTAMER MEDICINAL, what first need consideration is exactly how to extend its blood halflife.Current widely used mode generally has two kinds: one to be carry out molecular skeleton modification, such as protectiveness 2-position nucleotide modification (2-F, 2-OCH3, LNA, phosphate group) etc. to the Nucleotide of composition Nucleic acid aptamer molecules.Although the aptamer molecule modified through chemical group shows good anti-exonuclease characteristic, but the chemically modified of nucleotide backbone sometimes affects fit to the avidity of ligand molecular and the specificity of identification, the chemically modified nucleoside acid produced after aptamer molecule metabolism also may participate in the synthesis of human nucleic acid, there is potential harm.The second carries out cyclisation modification to aptamer molecule exactly.Because nuclease main in serum is exonuclease, the 5 ' end that cyclisation aptamer molecule does not expose and 3 ' end, the direct source of Exonucleolytic enzyme liberating is eliminated, and therefore aptamer molecule cyclisation is modified and can effectively be extended its serum stability.Cyclisation is fit owing to all adopting natural conventional Nucleotide, produces the risk having potential hazard product after avoiding fit metabolism.
The generation that current cyclisation is fit, mainly by the method that vitro enzyme connects, and utilizes generation cyclisation in plasmid body fit, there is efficiency low, the problem of complicated operation.How both to keep the Nucleic acid aptamer molecules filtered out through SELEX to identify and the characteristic combined with it ligand specificity, and improved again its body internal stability, also there is no system and ripe method.Therefore, develop a kind of rapidly and efficiently with the method for the fit preparation of the ring-type of low cost, the application of widening aptamer is had and important meaning.The present invention is directed to current APTAMER MEDICINAL Problems existing, develop a kind of can the fit cyclisation of high-level efficiency and low cost modification and the method for preparation.
Summary of the invention
For current APTAMER MEDICINAL Problems existing, the present invention establishes linear DNA aptamer molecule and carries out the method flow that design and preparation are modified in cyclisation, by rolling circle amplification (the rolling circle amplification improved, RCA) reaction produces cyclic DNA aptamer molecule, establish a kind of can efficiently fast and the aptamer molecule cyclisation of the Antineoplastic angiogenesis of low cost modify and design and preparation method's flow process.
The nucleotide sequence of DNA aptamer molecule is of paramount importance part in fit research, nucleotide sequence decide fit secondary structure and with the specificity of ligand molecular identification and binding ability, fitly directly cyclisation is carried out to linear molecule if most of, its original conformation can not be kept, thus lose or greatly weaken it and identify the ability with binding partner molecule.The present invention, according to the fit sequence of linear DNA filtered out through SELEX, determines through conformational analysis and core sequence, devises the fit sequence of cyclic DNA of the present invention.
DNA aptamer molecule cyclization reaction also exists cost height and inefficient problem at present, the present invention uses for reference RCA method, improve in the selection, amplification condition etc. of auxiliary sequencel design, reagent, establish a kind of can generation rapidly and efficiently, at low cost and comprise the fit techniqueflow of the ring-type of enriching loop-stem structure.Initial circular template molecule increase about 100,000 times can be made through twice RCA reaction.
The present invention writes a Chinese character in simplified form and is described as follows:
VE5a: through the linear fit sequence of the acted on VEGF165 that SELEX technology screening goes out;
CVE5a:VE5a aptamer molecule sequence does not add the sequence after the direct cyclisation of any change;
V5aE+: the linear aptamer molecule of VE5a adding AT joint sequence and restriction enzyme site sequence;
Sequence after p-V5aE+:V5aE+5 ' end phosphorylation;
The reverse complementary sequence of V5aE-:V5aE+;
The cyclisation aptamer molecule of cV5aE+:V5aE+;
The cyclisation aptamer molecule of cV5aE-:V5aE-, the reverse complementary sequence of cV5aE+;
The enzyme of LV5aE+:V5aE+ connects auxiliary sequencel;
The enzyme of LV5aE-:V5aE-connects auxiliary sequencel;
The enzyme of DV5aE+:cV5aE+ cuts auxiliary sequencel;
The enzyme of DV5aE-:cV5aE-cuts auxiliary sequencel;
ExoI: exonuclease I;
ExoIII: exonuclease III;
Forward primer in RV5aE+Pf: decoding for DTMF RCA reaction;
Reverse primer in RV5aE-Pr: decoding for DTMF RCA reaction;
TFpF: human tissue factor (Tissuefactor, TF) specific amplification forward primer;
TFpR: human tissue factor (Tissuefactor, TF) specific amplification reverse primer.
Above primer and oligonucleotide sequence are by Sangon Biotech's synthesis and purifying, and aseptic deionized water dilutes ,-20 DEG C of preservations.
In order to achieve the above object, technical scheme of the present invention is:
An aptamer molecule for Antineoplastic angiogenesis, its sequence is:
5 '-ACTTCCAGTCTATTCAATTGGGCCCGTCCGTATGGTGGGTGTGCGTGGCCACGAGT TTTAAAAAAAAAAACCAAGCTTTTTTTTGATCATGT-3 '; An AT joint and a nuclease action site are inserted in the position of this aptamer molecule (V5aE+) away from the core stem ring region from aptamer molecule and target molecule anti-vegf 165 specific combination, and wherein in AT joint, the number of A and T base is 8 respectively; Enzyme action site comprises DNA ligase enzyme vicinal point and DNA restriction endonuclease (RsaI) restriction enzyme site, position is between core stem ring region and AT joint, the molecule that cyclisation is modified is prepared to ring circulating reaction in a large number by ring, and cyclisation efficiency is detected by real time fluorescent quantitative RCA.
A preparation method for the aptamer molecule of Antineoplastic angiogenesis, comprises the steps: first to carry out phosphorylation and cyclization to linear aptamer molecule V5aE+, then enters ring to ring circulating reaction; Every secondary ring, to the reaction of ring circulating reaction experience two-wheeled, is often taken turns reaction and is followed successively by rolling circle amplification reaction, endonuclease reaction and cyclization.
The enzyme that described cyclization uses connects auxiliary sequencel LV5aE+ and is: 5 '-CCCAATTGAATAGACTGGAAGTACATGATCAAAAAAAAGCT-3 '; Or enzyme connects auxiliary sequencel LV5aE-and is: 5 '-AGCTTTTTTTTGATCATGTACTTCCAGTCTATTCAATTGGG-3 '.
The primer sequence that described rolling circle amplification reaction uses and enzyme connect auxiliary sequencel LV5aE+ and are: 5 '-CCCAATTGAATAGACTGGAAGTACATGATCAAAAAAAAGCT-3 '; Or enzyme connects auxiliary sequencel LV5aE-and is: 5 '-AGCTTTTTTTTGATCATGTACTTCCAGTCTATTCAATTGGG-3 '.
The enzyme that described endonuclease reaction uses is cut auxiliary sequencel DV5aE+ and is: 5 '-GAATAGACTGGAAGTACATGATC-3 '; Or enzyme cuts auxiliary sequencel DV5aE-:5 '-GATCATGTACTTCCAGTCTATTC-3 '.
Described cyclization condition is: connect auxiliary sequencel with 1-5 part enzyme mix linearly fit for the phosphorylation of 1 part of cyclisation, at Taq DNA ligase damping fluid (purchased from New EnglandBiolabs company, the U.S.) in, hatch 5 minutes, be then slowly cooled to room temperature for 95 DEG C; Add Taq DNA ligase (purchased from New England Biolabs company, the U.S.), hatch 4 minutes for 94 DEG C; Then complete 15 circulations according to following program: 94 DEG C 30 seconds, 45 DEG C 5 minutes; Last 95 DEG C of 15 minutes deactivation Taq DNA ligases; The DNA product exoI+exoIII of non-cyclisation is (purchased from New England Biolabs company, the U.S.) degraded: above-mentioned cyclization product mixtures, add Exo I and Exo III, hatch 2 hours 30 minutes for 37 DEG C, hatch 5 minutes deactivation excision enzymes, obtain cyclized DNA aptamer molecule for 80 DEG C.
The reaction conditions of described rolling circle amplification reaction is: 1 part of cyclized DNA molecule is connected auxiliary sequencel with the enzyme of 10-1000 part and mixes, join Bst DNA polymerase buffer liquid (purchased from New England Biolabs, the U.S.) in, add 200 ten thousand parts of dNTPs again (purchased from precious biotechnology company limited, Dalian), add Bst archaeal dna polymerase (purchased from NewEngland Biolabs, the U.S.) again, 65 DEG C 120 minutes; Hatch 20 minutes deactivation Bst archaeal dna polymerases for 80 DEG C.
The reaction conditions of described endonuclease reaction is: 1 part of RCA product and 100,000 parts of enzymes are cut auxiliary sequencel and joins RsaI restriction endonuclease damping fluid (purchased from New England Biolabs, the U.S.) in, add RsaI restriction enzyme again (purchased from New England Biolabs, the U.S.), hatch 3.0 hours for 37 DEG C; 65 DEG C of 20 minutes deactivation RsaI restriction endonucleases.
The invention has the beneficial effects as follows: effect detection is the key characterizing success or failure of the present invention, mainly comprise that amplification efficiency detects, cyclized DNA is fit Detection of Stability and biological activity assay.The present invention adopts quantitative real-time PCR to detect the serum stability of cyclisation amplification efficiency and cyclisation aptamer molecule; Adopt human umbilical vein epithelial cell (HUVEC) tissue factor (TF) mrna expression change experiment, the endocytobiology detecting the cyclisation aptamer molecule with anti-tumor neovascularization activity is active.
VEGF is a member in platelet derived growth factor superfamily, mainly comprises VEGF-A, placenta growth factor, VEGF-B, VEGF-C, VEGF-D and VEGF-E at present, and VEGF-A is wherein most important member.VEGF is by the conduction of its acceptor VEGFR mediate downstream signal, promote vascular endothelial cell proliferation, differentiation and maturation, inhibition apoptosis of vascular endothelial cell, induction of vascular smooth muscle cell migration, and promote that vascular smooth muscle cell is synthesized and secretion of MMPs, accelerate substrate degradation and chemotactic inflammatory cell etc., finally impel new angeogenesis and function maturation.The oxygen that the growth needs of cancer is a large amount of and nutritive substance, before diameter is about 1-2mm, cancerous tissue can obtain oxygen and nutritive substance by dispersion.After cancer diameter is more than 2mm, need new vessel to provide more oxygen and nutritive substance to maintain growth.When histanoxia, the cytokines such as HIF-1a (hypoxia-inducible factor-1 alpha) and MMP (matrix metalloproteinase) can promote the expression and secretion of VEGF.VEGF can not only induce cancer vasculogenesis, promotes cancer growth, also may participate in the formation of cancer cells resistance.Therefore VEGF is the most important target spot of cancer therapy drug.The target spot of the fit sequence that the present invention studies, namely important in VEGF family member VEGF165.
Described on end: the present invention establishes linear DNA aptamer molecule and carries out the method flow that design and preparation are modified in cyclisation, produce cyclic DNA aptamer molecule by the RCA reaction improved, establish and efficiently can prepare the fit method of cyclic DNA fast and at low cost.
Accompanying drawing explanation
Fig. 1 is the forecast analysis of the linear fit and fit secondary structure of cyclisation of anti-vegf 165 albumen; A is the result of VE5a being carried out to secondary structure prediction analysis, comprises three feature loop-stem structures of specific recognition target molecule VEGF165; B represents the result of cVE5a sequence being carried out to secondary structure prediction analysis; C represents the result of V5aE+ being carried out to secondary structure prediction analysis; D represents and carries out secondary structure prediction analytical results to cV5aE+.
Fig. 2 cyclic DNA aptamer molecule preparation flow figure; Comprise linear molecule phosphorylation and cyclisation two preparation step before circulation starts, after this experience twice RCA reaction, endonuclease reaction, cyclization, reaction process is simply efficient, and operation is easy to automatization.
Fig. 3 is embodiment of the present invention neutral line aptamer molecule cyclisation effect electrophoresis detection result.
Fig. 4 is the electrophoresis result of the embodiment of the present invention cyclized by treatment aptamer nucleic acid excision enzyme Detection of Stability; Wherein Fig. 4 A is the result of the fit VE5aE+ of linear DNA before and after exoI+exoIII and DNAseI process; Fig. 4 B is the result of DNA aptamer cVE5aE+ before and after exoI+exoIII and DNAse I process after cyclisation.
Fig. 5 is the electrophoresis result of the cyclized by treatment aptamer molecule of the embodiment of the present invention and target molecule VEGF165 protein binding effect detection; Wherein Fig. 5 A be VE5a (59nt) with VEGF121 and VEGF165 protein binding before and after electrophoresis result; 5B is the electrophoresis result before and after V5aE+ (92nt) is combined with VEGF121 and VEGF165; 5C is the electrophoresis result before and after cV5aE+ (92nt) is combined with VEGF165 and VEGF121.
Fig. 6 is the result that the embodiment of the present invention utilizes the fit cyclisation efficiency of RCA reaction bonded real-time fluorescence quantitative PCR detection of platform; Wherein Fig. 6 A is that the cV5aE+ of different concns is as quantitative PCR detection result during template; 6B is the linear standard curve between fluorescence obtained the maximum absorption and cV5aE+ concentration made according to Fig. 6 A result.
Fig. 7 is the fit real-time fluorescence quantitative PCR analytical results to the effect of human tissue factor (TF) expression amount of embodiment of the present invention VEGF165 and anti-vegf 165; Wherein Fig. 7 A is the quantitative PCR detection result of different concns VEGF165 to TF factor mrna expression amount promoter action; 7B is under the VEGF165 effect of 628pm, and different concns cV5aE+ and V5aE+ is by being combined with VEGF165 the inhibiting real-time fluorescence quantitative PCR detected result of TF factor mrna expression amount.
Embodiment
Embodiment 1
1, cyclisation aptamer molecule design
According to fit sequence VE5a (HijiriHasegawa, Oxford University Press (2007), the Nucleic AcidsSymposium Series No.51:399-400 of the VEGF165 gone out through SELEX technology screening; Biotechnol Lett (2008) 30:829-834), redesign can the fit sequence of circularized nucleic acid (V5aE+): its sequence is 5 '-ACTTCCAGTCTATTCAATTGGGCCCGTCCGTATGGTGGGTGTGCGTGGCCACGAGT TTTAAAAAAAAAAACCAAGCTTTTTTTTGATCATGT-3 '.As shown in 1A and 1B in Fig. 1, the linear fit V5aE gone out through SELEX technology screening rely on three feature loop-stem structures to the specific identification of protein target VEGF165 and combination (as solid line circle in Figure 1A identify), the direct cyclisation if do not modified to V5aE sequence, then identify that the feature loop-stem structure of target molecule sequence disappears (Figure 1B), it is necessary for therefore making number of base amendment to fit non-core sequence.
When designing cyclized DNA aptamer molecule sequence, the present invention adds AT joint sequence (in Fig. 1 C and 1D on the basis of linear fit sequence, dashed circle identifies) and restriction enzyme site and enzyme vicinal point (restriction enzyme site and enzyme vicinal point are at same position, pointed by Fig. 1 D, its position is away from three feature loop-stem structure districts); Molecule cV5aE+ after Fig. 1 C and 1D can find out the amended sequence V5aE+ of VE5a and cyclisation all remains it for identifying the feature loop-stem structure (solid circles identified) of target molecule VEGF165.
In order to improve the efficiency adopting RCA technology to prepare cyclic DNA aptamer molecule, the present invention devises enzyme and connects (LV5aE+ or LV5aE+) auxiliary sequencel; And according to the addition of RsaI restriction enzyme digestion sites (pointed by Fig. 1 D) in fit sequence, devising corresponding enzyme and cutting (DV5aE+ or DV5aE-) auxiliary sequencel.
2, the preparation of cyclisation aptamer molecule
The all flow processs of the present embodiment are as Fig. 2, first this flow process carries out phosphorylation and cyclization to V5aE+, then ring is entered to the ring RCA cycle stage, often take turns the twice RCA reaction of circulation experience, twice enzyme is cut, enzyme connects reaction and twice cyclization, from the cV5aE+ aptamer molecule RCA of ring-type, terminating to preparing the cV5aE+ molecule that makes new advances, often taking turns circulation and initial ring molecule can be made to increase 100,000 times, operation Cheng Liucheng is simple and be easy to automatization.
2.1 linear aptamer molecule phosphorylations
5 ' end of linear DNA aptamer molecule phosphorylation just must can carry out cyclization.Get 300nmol aptamer molecule (V5aE+) to join 5mL 10 × T4DNA Ligase damping fluid and (be purchased from precious biotechnology company limited, Dalian; Formula: the Tris-hydrochloric acid (pH7.6) of 660mM, the magnesium chloride of 66mM, the dithiothreitol (DTT) of 100mM, the ATP of 1mM) in, then add 10
4the T4 polynueleotide kinase (being purchased from precious biotechnology company limited, Dalian) of unit (U), final concentration is 1.5mM dATP (100mM is purchased from precious biotechnology company limited, Dalian), and distilled water complements to 50mL.Hatch 30 minutes for 37 DEG C, hatch 20 minutes deactivation T4 polynueleotide kinases for 65 DEG C, obtain 5 ' end phosphorylation after fit sequence (p-V5aE+).
2.2 linearly fit cyclizations
Get fit sequence (p-V5aE+) 18nmol after above-mentioned 5 ' end phosphorylation, connect auxiliary sequencel (LV5aE+) with 40nmol enzyme and mix, add distilled water to 10mL.First be heated to 95 DEG C 5 minutes, be then slowly cooled to room temperature.Add 3mL 10 × Taq DNA ligase damping fluid again (purchased from New England Biolabs company, the U.S.), the Taq DNA ligase of 8000U is (purchased from New England Biolabs company, the U.S.), distilled water supplies 30mL, hatch 4 minutes for 94 DEG C, then complete 15 circulations according to following program: 94 DEG C 30 seconds, 45 DEG C annealing 5 minutes; Last 95 DEG C of 15 minutes deactivation TaqDNA ligase enzymes.
The product exoI+exoIII of non-cyclisation degrades.Get the above-mentioned cyclization product mixtures of 30mL, add 3.0mL 10 × Exo III damping fluid (purchased from New England Biolabs company, the U.S.; Formula: the Bis-Tris-propane-hydrochloric acid of 10mM, the magnesium chloride of 10mM, the dithiothreitol (DTT) of 1mM, PH 7.0), and then add the Exo III (Exo I and Exo III all purchased from New England Biolabs company, the U.S.) of the Exo I of 4500U and 1.5 × 105U; Distilled water complements to 30mL, hatches 2.5h for 37 DEG C, hatches 15 minutes deactivation excision enzymes for 80 DEG C, obtains cyclized DNA aptamer molecule (cV5aE+).
2.3 rolling circle amplification reactions 1
30pmol ring-shaped DNA molecule (cV5aE+) is joined 6mL 10 × Bst DNA polymerase buffer liquid (purchased from New England Biolabs company, the U.S.; Formula: the Tris-hydrochloric acid of 20mM, the ammonium sulfate of 10mM, the Repone K of 10mM, the magnesium sulfate of 2mM, the Triton X-100 of 0.1%, PH 8.8) in, add the dNTPs (10mM of 60 μm of ol again, be purchased from precious biotechnology company limited, Dalian), the RCA primer LV5aE+ of 15nmol, 4UBst archaeal dna polymerase is (purchased from New England Biolabs company, the U.S.), 8% glycerine (volume percent), distilled water complements to 60mL.65 DEG C 120 minutes, hatch 20 minutes deactivation Bst archaeal dna polymerases for 80 DEG C, obtain comprise multiple V5aE-molecule repeat long-chain DNA sequence dna.In the RCA reaction of this step, amplimer adopts enzyme to connect auxiliary sequencel LV5aE+.
2.4 endonuclease digestion reactions 1
The enzyme of 1pmol RCA product and 100nmol is cut auxiliary sequencel (DV5aE-) join 3.0mL10 × RsaI limit restriction endonuclease damping fluid (purchased from New England Biolabs company, the U.S.; Formula: the Tris-acetic acid of 20mM, the Potassium ethanoate of 50mM, the magnesium acetate of 10mM, the dithiothreitol (DTT) of 1mM, PH 7.9), then add the RsaI restriction enzyme of 104U (purchased from New England Biolabs company, the U.S.), distilled water complements to 30mL.Hatch 3.0 hours for 37 DEG C.65 DEG C of 20 minutes deactivation RsaI restriction endonucleases.Because RCA product and DV5aE-form local double-stranded DNA, the linear DNA fragment (V5aE-) with linear aptamer molecule V5aE+ reverse complemental can be formed by RsaI enzyme inscribe.
2.5V5aE-cyclization 1
The V5aE-sample of 18nmol is mixed with the LV5aE-of 40nmol, adds distilled water to 10mL, be heated to 95 DEG C, be then slowly cooled to room temperature.Add 3.0mL10 × Taq DNA ligase damping fluid (purchased from New England Biolabs company, the U.S.; Formula: the Tris-hydrochloric acid of 20mM, the Potassium ethanoate of 25mM, the magnesium acetate of 10mM, the NAD of 1mM, the two sulfuric acid sugar alcohols of 10mM, the Triton X-100 of 0.1%, pH 7.6), the TaqDNA ligase enzyme of 8000U is (purchased from New England Biolabs company, the U.S.) in reaction system, add distilled water and supply 30mL, hatch 4 minutes for 94 DEG C, then complete 15 circulations according to following program: 94 DEG C 30 seconds, 45 DEG C annealing 5 minutes; Last 95 DEG C of 15 minutes deactivation TaqDNA ligase enzymes.Obtain cyclized DNA fragment cV5aE-.
The DNA product exoI+exoIII of non-cyclisation degrades, and system is with 2.2 steps.
2.6 rolling circle amplification reactions 2
The cV5aE-of 600pmol is joined 6mL 10 × Bst DNA (NEB) polymerase buffer (purchased from New England Biolabs company, the U.S.) in, add the dNTPs (10mM of 60 μm of ol again, purchased from precious biotinylated biomolecule Engineering Co., Ltd, Dalian), the RCA amplimer LV5aE-of 15nmol, 4000U Bst archaeal dna polymerase is (purchased from New EnglandBiolabs company, the U.S.), 8% glycerine (volume percent), distilled water complements to 60mL.65 DEG C 120 minutes, hatch 20 minutes deactivation Bst archaeal dna polymerases for 80 DEG C, react complete acquisition comprise multiple V5aE+ molecule repeat long-chain DNA sequence dna.
2.7 endonuclease digestion reactions 2
Get the product of 1pmolRCA reaction 2, cut auxiliary sequencel (DV5aE+) with the enzyme of 100nmol and mix, join in 3.0mL 10 × RsaI restriction enzyme damping fluid, then add 10
4the RsaI restriction enzyme (purchased from New England Biolabs company, the U.S.) (buying manufacturer) of unit, distilled water complements to 30mL.Hatch 3.0 hours for 37 DEG C.Long-chain RCA product enzyme can be cut into single linear aptamer molecule (V5aE+) fragment.
65 DEG C of 20 minutes inactivation RsaI restriction endonucleases.
2.8V5aE+ aptamer molecule cyclization 2
Get the V5aE+ product 18nmol in 2.7 steps, connect auxiliary sequencel (LV5aE+) with 40nmol enzyme and mix, add distilled water to 10mL.First be heated to 95 DEG C, be then slowly cooled to room temperature.Add 3mL 10 × Taq DNA ligase damping fluid (purchased from New EnglandBiolabs company, the U.S.), the Taq DNA ligase of 8000U is (purchased from New EnglandBiolabs company, the U.S.), distilled water supplies 30mL, hatch 4 minutes for 95 DEG C, then complete 15 circulations according to following program: 94 DEG C 30 seconds, 45 DEG C annealing 5 minutes; Last 95 DEG C of 15 minutes deactivation TaqDNA ligase enzymes.
The product exoI+exoIII of non-cyclisation degrades, and operation, with 2.2 steps, can obtain the cyclisation aptamer molecule (cV5aE+) of amplification about 100,000 times.
3, effect detection
3.1 cyclisation rolling circle amplification product detect
The present invention devises decoding for DTMF RCA reaction and carries out the fit RCA product detection of cyclisation: by cyclisation product after exoI+exoIII digestion, linear DNA fragment is digested, only surplus cyclic products.
Get 50fmol ring-shaped DNA molecule and join 2 μ L BstDNA polymerase buffers (purchased from New England Biolabs company, the U.S.) in, add 15fmol RV5aE+pF again, (10mM, purchased from precious biotinylated biomolecule engineering corporation for 15fmol RV5aE-pR, 0.3nmol dNTPs, Dalian), 1.6U Bst archaeal dna polymerase (purchased from New England Biolabs company, the U.S.), adds distilled water to 20 μ L.65 DEG C 120 minutes.3% agarose gel electrophoresis detects, and result such as Fig. 3, Fig. 3 are embodiment of the present invention neutral line aptamer molecule cyclisation effect electrophoresis detection result.In Fig. 3, swimming lane 1 carries out RCA reaction result again for V5aE+ (5 ' holds non-phosphorylation) after the ligation of Taq DNA ligase, because 5 ' end does not carry out phosphorylation, therefore can not by cyclisation, and RCA also just occurs without scalariform band.Swimming lane 2 be ring-type aptamer molecule after p-V5aE+ success cyclisation through the reacted result of RCA, to have specific to decoding for DTMF RCA product scalariform band occur.
3.2 cyclisation Efficiency testing
In order to detect linearly fit cyclisation effect and cyclisation efficiency, design a series of decoding for DTMF amplification RCA reaction.Get the cV5aE+ molecule (0,25,50 of different amount, 100,150,200,250,300fmol) do RCA typical curve, make the fit sample of ring-type (sample (sample 1) that embodiment 2 step 2.2 purifying is good and the good sample of 2.8 purifying carry out concentration dilution 10 simultaneously
4fluorescence real-time quantitative RCA doubly (sample 2)) reacts, and reacts at real-time quantitative fluorescence PCR instrument (StepOne
tMplus, Applied Biosystems, the U.S.) in carry out, reaction system 20 μ L:2.0 × BstDNA polymerase buffer, 0.5 μ L 20 × SYBR Green I dye solution is (purchased from Shanghai Shan Jing molecular biosciences Science and Technology Ltd., Shanghai), the RV5aE+pF of 0.3pmol (synthesizes from Sangon Biotech (Shanghai) Co., Ltd., Shanghai), the RV5aE-pR of 0.3pmol (synthesizes from Sangon Biotech (Shanghai) Co., Ltd., Shanghai), 30nmol dNTPs is (purchased from precious biotinylated biomolecule Engineering Co., Ltd, Dalian), 1.6U Bst archaeal dna polymerase is (purchased from New England Biolabs company, the U.S.), distilled water supplies 20 μ L.65 DEG C are increased 90 minutes.Result data adopts StepOne Software v2.1 software analysis.Result such as Fig. 6, Fig. 6 are the result that the embodiment of the present invention utilizes the fit cyclisation efficiency of RCA reaction bonded real-time fluorescence quantitative PCR detection of platform; Fig. 6 A is in the scope of 0-10nmol, the amplification curve of the RCA template real-time quantitative PCR reaction of different concns.From figure, the cV5aE+ sample obtained the maximum absorption that concentration is low is lower, and the required cycle number that absorption value reaches quick ascent stage is more, has good linear relationship after proving cV5aE+ and RCA amplification between fluorescent absorption value parameter.Fig. 6 B be RCA reaction cV5aE+ template concentrations and fluorescence obtained the maximum absorption through statistics analysis software package (statistical product and service solution software package, Statistical Product and Service Solutions, SPSS) matching, there is good linear relationship: (y is obtained the maximum absorption logarithm to y=0.708x+2.708, x is the logarithm of cyclisation aptamer concentrations (nM) value, R2=0.966).After calculating amplification according to linear equation, ring molecule adds about 0.75 × 10 than before amplification
5doubly.
3.3 cyclisation aptamer molecule excision enzyme stability
After getting 20 μ L cyclizations, fit and linear fit sample adds 2.5 μ L10 × Exo III damping fluids (purchased from New England Biolabs company, the U.S.) respectively, then adds the Exo III of Exo I and 100U of 3U; Distilled water complements to 25 μ L, hatches 2.5h for 37 DEG C, hatches 15 minutes deactivation excision enzymes for 80 DEG C.
Cyclisation is fit and linear fit sample also joins in the damping fluid of 10 × DNase I of 2.0 respectively (purchased from New England Biolabs company, the U.S.), add the DNase I of 22.5U again (purchased from New England Biolabs company, the U.S.), distilled water supplies 20 μ L, hatches 2.0h for 25 DEG C.
Reaction product with 8% sex change PAGE gel (0.5mg/L GoldView, 0.5 × tbe buffer liquid) electrophoresis detection, deposition condition: 150V, 30 minutes.Result such as Fig. 4, Fig. 4 are the electrophoresis result of the embodiment of the present invention cyclized by treatment aptamer nucleic acid excision enzyme Detection of Stability; Fig. 4 A: linear V5aE+ (92nt) sample of swimming lane 1, swimming lane 2 and 3 is the V5aE+ sample through exoI+exoIII process, and 4 is DNase I process V5aE+ sample.According to Fig. 4 A result, linear V5aE+ is fit, and sample is all degradable by exoI+exoIII; Fig. 4 B: swimming lane 1 is the fit sample of cV5aE+, swimming lane 2 and 3 is the cV5aE+ sample through exoI+exoIII process, and swimming lane 4 is the cV5aE+ sample of DNase I process.M is 20-500bp molecular weight standard, and the 3rd band is 100bp.Band still can be had to occur from the fit sample electrophoresis of Fig. 4 B, cV5aE+, prove fit can the degraded of effectively anti-exoI+exoIII after cyclisation.From Fig. 4 result, and no matter linear or ring-type is fit, all effectively can't resist non-specific DNA endonucleases.
3.4 detect with ligand molecular VEGF165 bonding force
Gel retardation assasy (EMSA) is adopted to carry out.Respectively cV5aE+ (4pmol) good for purifying, VE5a (4pmpl), V5aE+ (4pmol) are joined 4 μ L 1 × TBSE binding buffer liquid (10mM Tris/HCl, pH 7.0,100mM NaCl, 0.05mM EDTA) in, 95 DEG C are heated 5 minutes, are then progressively cooled to 25 DEG C with the speed of 2 DEG C/min.Then add 100ng VEGF165 or VEGF121 and hatch 1.0h at 25 DEG C, add 2 μ L 6 × loadingbuffer, join in the non-denaturing polyacrylamide gel (purchased from sigma company, the U.S.) of 15%, then run 40 minutes under 200V voltage.Electrophoresis is complete, and dye GoldView dyestuff (purchased from match Parkson, Beijing gene engineering company limited, Beijing) 30min, projects instrument (upper sea route sun Instrument Ltd., Shanghai) check by ultraviolet, and photographic analysis.The results are shown in Figure 5, in figure, 5A is the fit (VE5a through SELEX screening, 59nt) with protein target VEGF165 and non-target molecules VEGF121 mixing electrophorogram, M is 20-500bp molecular weight standard, swimming lane 1 is the electrophoresis result of simple VE5a molecule, swimming lane 2 is the result that VE5a mixes with non-target molecules VEGF121 albumen, and swimming lane 3 hatches rear electrophoresis result for VE5a and VEGF165 mixes; As can be seen from Figure 5A, after VE5a target molecule VEGF165, electrophoretic band is a bit delayed, and is not combined with non-target molecules VEGF121, electrophoretic band invariant position; Fig. 5 B is V5aE+ (92nt) and target molecule VEGF165 and non-target molecules VEGF121 mixing rear electrophoresis figure, swimming lane 1 is V5aE+ molecular sample, swimming lane 2 is the result that V5aE+ and non-target molecules VEGF121 albumen are hatched jointly, and swimming lane 3 hatches rear result jointly for V5aE+ and protein target VEGF165; Fig. 5 C is cyclisation aptamer molecule cV5aE+ (92nt) and target molecule VEGF165 and non-target molecules VEGF121 mixing rear electrophoresis figure, swimming lane 1 hatches rear result jointly for cV5aE+ and protein target VEGF165, swimming lane 2 is cV5aE+ molecular sample, and swimming lane 3 is the result that cV5aE+ and non-target molecules VEGF 121 albumen are hatched jointly.From result, no matter linearly fit fit in binding buffer liquid with ring-type, after jointly hatching with VEGF165, electrophoretic band a bit delayed (swimming lane A3, B3, C1), and after jointly hatching with non-target molecules albumen VEGF121, electrophoretic band position does not change compared with simple aptamer molecule (swimming lane A2, B2, C3).After aptamer molecule is combined with VEGF165 by analysis, electrophoretic band position is not consistent with its molecular weight, major cause is that this experiment adopts Native PAGE electrophoresis, molecular weight analyte, protein structure, the institute factor such as electrically charged determines jointly can pillar location, and after DNA aptamer molecule combines with it, conformation also will change, although so delayed, inconsistent with its molecular weight relation.
As seen from the figure: after VE5a, V5aE+, cV5aE+ and VEGF165 combine, electrophoretic band is delayed all to some extent compared with simple DNA sample, and it is constant in conjunction with rear electrophoresis pillar location with VEGF121, prove that these aptamer molecules all can specific recognition and binding partner albumen VEGF165, and nonrecognition with in conjunction with non-target protein VEGF121.
3.5 intracellular biological Function detection
Human umbilical vein epithelial cell (HUVEC) tissue factor (TF) expression amount is adopted to prevent experiment to be verified: VEGF165 can promote the expression of human tissue factor (TF) factor, anti-vegf 165 is fit in vivo with it combine after, then prevented the rising of TF factor expression amount.
The 1640 culture medium culturing 4hs of HUVEC containing 1% serum, then the VEGF165 of different concns is added respectively (purchased from Sino Biological Inc., Beijing) process 1.0h (0.172nM, 0.328nM, 0.626nM, 1.25nM, 2.39nM), with not being the HUVEC of any process as negative control.The extraction of total serum IgE adopts Trizo method to carry out.The mrna expression level real-time quantitative fluorescence PCR (StepOne of human tissue factor (TF) factor
tMplus, Applied Biosystems, the U.S.) method determination and analysis.(Beijing Zhuan Meng is biological, Beijing for reaction system 20 μ L:1.0 × PCR reaction mixture; Comprise archaeal dna polymerase, SYBRGreen I dyestuff, dNTPs, reaction buffer etc.), the TFpF (being synthesized by Sangon Biotech (Shanghai) Co., Ltd., Shanghai) of the TFpF of 0.2pmol, 0.2pmol, distilled water supplies 20 μ L.Internal reference is GAPDH gene, and data acquisition StepOne Software v2.1 software analysis, the relative untreated samples of mNRA expression level of TF has done stdn.Result such as Fig. 7 A, Fig. 7 are the fit real-time fluorescence quantitative PCR analytical results to the effect of TF mrna expression amount of embodiment of the present invention VEGF165 and anti-vegf 165.What Fig. 7 adopted is relative expression quantity analytical model.Wherein Fig. 7 A be different concns VEGF165 to human tissue factor (TF) expression amount exercising result, to the promoter action of TF expression amount maximum (in Fig. 7 A circle mark) when wherein VEGF165 concentration is 626pM.
VEGF165 has maximum facilitation effect when 626pM to the expression of TF factor mRNA, and namely the present invention verifies that ring-type is fit active at intracellular biological at this concentration.
The 1640 culture medium culturing 4hs of HUVEC cell containing 1% serum, then 626pMVEGF165 is added, then the linearly fit (V5aE+ of different concns is added, 3.0nm-48nM) and the fit (cV5aE+ of ring-type, 3.0nM-48nM), effect 1h, adopts the HUVEC not doing any process as negative control.The extraction of total serum IgE adopts Trizol method to carry out.TF mrna expression level real-time quantitative fluorescence PCR (StepOne
tM) determination and analysis, reaction system 20 μ L:1.0 × PCR reaction mixture (being purchased from Beijing Zhuan Meng biological, Beijing), the TFpF of the TFpF of 0.2pmol, 0.2pmol, distilled water supplies 20 μ L.Internal reference is GAPDH gene, and data acquisition StepOne Software v2.1 software analysis, the relative untreated samples of TF mNRA expression level has done stdn.Result as Fig. 7 B, Fig. 7 B be linear and cyclic DNA fit by being combined the effect that inhibit it to TF factor mrna expression amount with VEGF165.What ellipse identified is that cV5aE+ aptamer molecule has the concentration of maximum suppression effect to TF factor mrna expression, is 6nM; What positive round identified is that linear fit V5aE+ has the concentration of maximum suppression effect to TF factor mrna expression, is 12nM.Dotted ellipse mark be do not add aptamer molecule, the expression amount of TF factor mRNA when only adding 626pM VEGF165.Result show cyclisation modify after fitly still remain biologic activity, and the linear fit biologic activity of more non-cyclisation increases (improving about 1 times).
Claims (1)
1. a preparation method for the cyclisation aptamer molecule of Antineoplastic angiogenesis, this cyclisation aptamer molecule is by can based on the fit V5aE+ of circularized nucleic acid, and this can the sequence of the fit V5aE+ of circularized nucleic acid be:
5 '-ACTTCCAGTCTATTCAATTGGGCCCGTCCGTATGGTGGGTGTGCGTGGCCACGAGT TTTAAAAAAAAAAACCAAGCTTTTTTTTGATCATGT-3 '; This can insert an AT joint and a nuclease action site in the position of the fit V5aE+ of circularized nucleic acid away from the core stem ring region from aptamer molecule and target molecule VEGF165 specific combination, and wherein in AT joint, the number of A and T base is 8 respectively; Enzyme action site comprises DNA ligase enzyme vicinal point and DNA restriction endonuclease RsaI restriction enzyme site, position is between core stem ring region and AT joint, the molecule that cyclisation is modified is prepared to ring circulating reaction in a large number by ring, and cyclisation efficiency is detected by real time fluorescent quantitative RCA; It is characterized in that comprising the steps:
First phosphorylation and cyclization are carried out to V5aE+, then ring is entered to the ring RCA cycle stage, often take turns the twice RCA reaction of circulation experience, twice enzyme is cut, enzyme connects reaction and twice cyclization, from the cV5aE+ aptamer molecule RCA of ring-type, the cV5aE+ molecule made new advances to preparation terminates;
S1: linear aptamer molecule phosphorylation
5 ' end of linear DNA aptamer molecule phosphorylation just must can carry out cyclization; Get 300nmol aptamer molecule V5aE+ to join in 5mL 10 × T4DNA Ligase damping fluid, then add 10
4the T4 polynueleotide kinase of U, distilled water complements to 50Ml, and final concentration is 1.5mM dATP; Hatch 30 minutes for 37 DEG C, hatch 20 minutes deactivation T4 polynueleotide kinases for 65 DEG C, obtain 5 ' end phosphorylation after fit sequence p-V5aE+;
S2: linearly fit cyclization
Get the fit sequence p-V5aE+18nmol after above-mentioned 5 ' end phosphorylation, connect auxiliary sequencel LV5aE+ with 40nmol enzyme and mix, add distilled water to 10Ml; First be heated to 95 DEG C 5 minutes, be then slowly cooled to room temperature; Add 3mL 10 × Taq DNA ligase damping fluid again, the Taq DNA ligase of 8000U, distilled water supplies 30mL, hatches 4 minutes for 94 DEG C, then completes 15 circulations according to following program: 94 DEG C 30 seconds, 45 DEG C annealing 5 minutes; Last 95 DEG C of 15 minutes deactivation TaqDNA ligase enzymes;
The product exoI+exoIII of non-cyclisation degrades; Get the above-mentioned cyclization product mixtures of 30mL, add 3.0mL 10 × Exo III damping fluid, and then add the Exo I and 1.5 × 10 of 4500U
5the Exo III of U; Distilled water complements to 30mL, hatches 2.5h for 37 DEG C, hatches 15 minutes deactivation excision enzymes for 80 DEG C, obtains cyclized DNA aptamer molecule cV5aE+;
S3: rolling circle amplification reaction 1
Joined by 30pmol ring-shaped DNA molecule cV5aE+ in 6mL 10 × Bst DNA polymerase buffer liquid, then add the dNTPs of 60 μm of ol, the RCA primer LV5aE+ of 15nmol, 4U Bst archaeal dna polymerase, 8% glycerine, distilled water complements to 60Ml; 65 DEG C 120 minutes, hatch 20 minutes deactivation Bst archaeal dna polymerases for 80 DEG C, obtain comprise multiple V5aE-molecule repeat long-chain DNA sequence dna, V5aE-is the linear DNA fragment with V5aE+ reverse complemental; In the RCA reaction of this step, amplimer adopts enzyme to connect auxiliary sequencel LV5aE+;
S4: endonuclease digestion reaction 1
The enzyme of 1pmol RCA product and 100nmol is cut auxiliary sequencel DV5aE-to join 3.0mL10 × RsaI and limit restriction endonuclease damping fluid, then add 10
4the RsaI restriction enzyme of U, distilled water complements to 30mL; Hatch 3.0 hours for 37 DEG C, 65 DEG C of 20 minutes deactivation RsaI restriction endonucleases, because RCA product and DV5aE-form local double-stranded DNA, the linear DNA fragment V5aE-with linear aptamer molecule V5aE+ reverse complemental can be formed by RsaI enzyme inscribe;
The cyclization 1 of S5:V5aE-
The V5aE-sample of 18nmol is mixed with the LV5aE-of 40nmol, add distilled water to 10mL, be heated to 95 DEG C, be then slowly cooled to room temperature, add 3.0mL10 × Taq DNA ligase damping fluid, the TaqDNA ligase enzyme of 8000U is in reaction system, add distilled water and supply 30mL, hatch 4 minutes for 94 DEG C, then complete 15 circulations according to following program: 94 DEG C 30 seconds, 45 DEG C annealing 5 minutes; Last 95 DEG C of 15 minutes deactivation TaqDNA ligase enzymes, obtain cyclized DNA fragment cV5aE-;
The DNA product exoI+exoIII of non-cyclisation degrades, and system is with S2 step;
S6: rolling circle amplification reaction 2
The cV5aE-of 600pmol is joined in 6mL 10 × Bst DNA polymerase buffer liquid, add the dNTPs of 60 μm of ol again, the RCA amplimer LV5aE-of 15nmol, 4000U Bst archaeal dna polymerase, 8% glycerine, distilled water complements to 60Ml, 65 DEG C 120 minutes, hatch 20 minutes deactivation Bst archaeal dna polymerases for 80 DEG C, react the long-chain DNA sequence dna that complete acquisition comprises the repetition of multiple V5aE+ molecule;
S7: endonuclease digestion reaction 2
Get the product of 1pmolRCA reaction 2, cut auxiliary sequencel DV5aE+ with the enzyme of 100nmol and mix, join in 3.0mL 10 × RsaI restriction enzyme damping fluid, then add 10
4the RsaI restriction enzyme of unit, distilled water complements to 30Ml, hatches 3.0 hours for 37 DEG C, long-chain RCA product enzyme can be cut into single linear aptamer molecule V5aE+ fragment;
65 DEG C of 20 minutes inactivation RsaI restriction endonucleases;
S8:V5aE+ aptamer molecule cyclization 2
Get the V5aE+ product 18nmol in S7 step, connect auxiliary sequencel LV5aE+ with 40nmol enzyme to mix, add distilled water to 10Ml, be first heated to 95 DEG C, be then slowly cooled to room temperature, add 3mL10 × Taq DNA ligase damping fluid, the Taq DNA ligase of 8000U, distilled water supplies 30mL, hatches 4 minutes for 95 DEG C, then complete 15 circulations according to following program: 94 DEG C 30 seconds, 45 DEG C annealing 5 minutes; Last 95 DEG C of 15 minutes deactivation TaqDNA ligase enzymes;
The product exoI+exoIII of non-cyclisation degrades, and operates same S2 step, can obtain the cyclisation aptamer molecule cV5aE+ of amplification 100,000 times;
The enzyme that above-mentioned each cyclization uses connects auxiliary sequencel LV5aE+ and is: 5 '-CCCAATTGAATAGACTGGAAGTACATGATCAAAAAAAAGCT-3 '; Enzyme connects auxiliary sequencel LV5aE-: 5 '-AGCTTTTTTTTGATCATGTACTTCCAGTCTATTCAATTGGG-3 ';
The primer sequence that above-mentioned each rolling circle amplification reaction uses and enzyme connect auxiliary sequencel LV5aE+ and are: 5 '-CCCAATTGAATAGACTGGAAGTACATGATCAAAAAAAAGCT-3 '; Enzyme connects auxiliary sequencel LV5aE-: 5 '-AGCTTTTTTTTGATCATGTACTTCCAGTCTATTCAATT GGG-3 ';
The enzyme that above-mentioned each endonuclease reaction uses is cut auxiliary sequencel DV5aE+ and is: 5 '-GAATAGACTGGAAGTACATGATC-3 '; Enzyme cuts auxiliary sequencel DV5aE-: 5 '-GATCATGTACTTCCAGTCTATTC-3 '.
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