CN102955047A - Biomolecule affinity constant determination method based on DNA (Deoxyribonucleic Acid) origami - Google Patents
Biomolecule affinity constant determination method based on DNA (Deoxyribonucleic Acid) origami Download PDFInfo
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Abstract
本发明公开了一种基于DNA折纸的生物分子的亲和常数测定方法。所述生物分子包括能够彼此结合与解离的反应物Ⅰ和反应物Ⅱ，该方法包括（1）将反应物Ⅰ连接于DNA折纸订书钉链末端，然后和脚手架链进行自组装形成DNA折纸；（2）加入反应物Ⅱ，反应达到平衡后用原子力显微镜扫描成像收集数据，计算出反应物I和反应物Ⅱ复合物的浓度[AbAg]，游离反应物Ⅰ的浓度[Ag]以及游离反应物Ⅱ的浓度[Ab]；（3）将所得数据代入公式K=[AbAg]/[Ag][Ab]计算得到所述反应物Ⅰ和反应物Ⅱ的亲和常数K。该方法从分子水平精确计算所述生物分子的亲和常数，具有高效、重复性好和样品用量少的优势。The invention discloses a DNA origami-based biomolecular affinity constant determination method. The biomolecules include reactant I and reactant II capable of combining and dissocating with each other, and the method includes (1) connecting reactant I to the end of a DNA origami staple chain, and then self-assembling with the scaffold chain to form DNA origami ; (2) Add reactant Ⅱ, after the reaction reaches equilibrium, use atomic force microscope to scan and image to collect data, and calculate the concentration of the compound of reactant I and reactant Ⅱ [AbAg], the concentration of free reactant Ⅰ [Ag] and the free reaction (3) Substituting the obtained data into the formula K=[AbAg]/[Ag][Ab] to calculate the affinity constant K of the reactant I and reactant II. The method accurately calculates the affinity constant of the biomolecule at the molecular level, and has the advantages of high efficiency, good repeatability and less sample consumption.
Description
Technical field
The invention belongs to the detection method field, particularly a kind of assay method of the biomolecule affinity costant based on DNA paper folding.
Background technology
The affinity of antibody (affinity) refers to the firm degree that antigen is combined with antibody.The association reaction of the larger expression antibody of affinity is faster, and antigen antibody complex more is difficult for dissociating.Affinity of antibody is binding site and the non-covalent gravitation between the corresponding antigens determinant and the repulsion sum of antibody, and it is the important indicator of estimating the antibody quality.The size of affinity of antibody represents with affinity costant, and affinity constant (K) is the equilibrium constant of antigen/antibody association reaction.
At present, the method for mensuration affinity costant has multiple, as: thiocyanate elution method, urea elution method, ELISA method, biology sensor method, surface plasma resonance method and competition combined techniques etc.The said determination method mainly is based upon on the statistics basis of macroscopic view, at present also without the assay method on the single molecules level.
The mensuration of affinity costant is based on following in conjunction with the dissociation theorem, shown in following chemical formula:
When reaction reaches balance, shown in the following row mathematical expression of its affinity costant computing method:
K=[AbAg]/[Ag][Ab]
Wherein, [Ag] represents free antigen concentration, and [Ab] represents free antibodies concentration, and [AbAg] represents antigen antibody complex concentration, and K is affinity costant.
The method of traditional mensuration affinity costant can not directly be measured affinity costant from above-mentioned mathematical expression, because the free antigen in can't determining to react and free antibodies concentration, so need transformation for mula to measure.The common setting of a tradition assay method wherein concentration of component is far smaller than an other concentration of component, is far smaller than antibody concentration such as antigen concentration.Like this, reaction just can think that the antibody total concentration is free antibodies concentration when reaching balance, brings the formula calculating after the conversion into.The method of using at present more mensuration biomolecule affinity costant is mainly enzyme-linked immunosorbent assay (ELISA) and surface plasma body resonant vibration method (SPR).ELISA measures the OD value to calculate affinity costant, and SPR is calculating K
b(association rate constant) and K
d(dissociation rate constant) measures affinity costant K, and the ground unrest of these two kinds of methods is comparatively speaking than all larger, and the mensuration process is also relatively complicated.Therefore, traditional assay method not only needs to consume a large amount of antigen, antibody, and the condition that experiment is set is more, and step is more loaded down with trivial details and complicated, and the error of introducing is also relatively large.
DNA paper folding art is the nanometer technology that obtains fast development recent years, utilizes a long single stranded DNA (the framing scaffold chain is generally M13mp18) and more than 200 oligonucleotide chain (chain of stapling together) the short chain self assembly of mutually pairing to form.By designing different oligonucleotide chains, can make DNA be assembled into different nanostructureds, its structure have pattern controlled and the coding governed feature.Because an oligonucleotides chain end can be modified by different kinds of molecules, so the reaction site on DNA paper folding interface has accurately controlled advantage.Atomic force microscope (AFM) has nano level resolution, change the situation that just can accurately judge reactions change by the pattern that detects ad-hoc location in the paper folding, whether occur such as reaction, and the reaction velocity speed etc., can be at single molecules level research biomolecule pattern and intermolecular interaction.
Summary of the invention
Therefore, the technical problem to be solved in the present invention can't accomplish to measure at single molecules level for existing biomolecule affinity costant assay method exactly, and the condition that experiment is set is more, complex steps and complexity, the error of introducing is relatively large problem also, utilize DNA paper folding to have accurately controlled advantage and atomic force microscope (AFM) advantage that can on reaction interface, directly observe single antigen-antibody molecular complex of reaction site, a kind of affinity costant assay method of the biomolecule based on DNA paper folding is provided.
For solving the problems of the technologies described above, one of technical scheme that the present invention takes is: a kind of affinity costant assay method of the biomolecule based on DNA paper folding, wherein said biomolecule comprises reactant I and the reactant II that can be bonded to each other and dissociate, and this assay method may further comprise the steps:
(1) the reactant I is connected in DNA paper folding staple chain end, then forms DNA paper folding with the self assembly of framing scaffold chain;
(2) add the reactant II, after reaching balance, reaction utilizes the afm scan imaging collection data, calculate the concentration [AbAg] of reactant I and reactant II compound, the concentration [Ab] of the concentration [Ag] of free reactant I and free reactant II;
(3) with the following formula of step (2) institute's value substitution, K=[AbAg]/[Ag] [Ab], calculate the affinity costant K of described reactant I and reactant II.
Wherein said biomolecule be can occur between the unimolecule of this area routine in conjunction with and a pair of biomolecule of dissociating.Described biomolecule preferably comprises reactant I and the reactant II of specific binding with it, described reactant I and with it the reactant II of specific binding preferably comprise in following each component one or more groups: antigen and antibody, biotin and Avidin, aptamer and target molecule thereof and part and acceptor.Wherein also comprise other known materials that specific binding can occur and have affinity costant, these are all within protection scope of the present invention.Such as: the materials such as protein, polysaccharide and polypeptide that can combine with DNA aptamers or RNA aptamers.Reactant I of the present invention and reactant II are preferably digoxin and DigiTAb.
Wherein step (1) is described is connected in DNA paper folding staple chain end with the reactant I, and then the method with framing scaffold chain self assembly formation DNA paper folding is the conventional DNA paper folding in this area self-assembling method.Wherein said self-assembling method preferably may further comprise the steps: the staple chain that will modify reactant I mixes the rear damping fluid that adds with staple chain and the framing scaffold chain of unmodified, put into the PCR instrument, after DNA paper folding self assembly occurs under certain annealing temperature, method with ultrafiltration is removed unnecessary staple chain, namely gets described DNA paper folding.Wherein said damping fluid is the conventional damping fluid in this area, is preferably 1 * TAE/Mg
2+Buffer solution system.Wherein said annealing temperature preferably is to be annealed to 20 ℃ from 95 ℃, and annealing speed preferably is 0.1 ℃/10 seconds; Wherein said framing scaffold chain is genomic DNA preferably, is preferably M13mp18DNA.Framing scaffold chain and staple strand are 1:10～1:5 preferably in molar ratio, are preferably 1:10.Described framing scaffold chain is a kind of commercially produced product of maturation, also can adopt conventional gene engineering method to obtain.
Wherein said DNA paper folding is the conventional described DNA paper folding in this area.The pattern of described DNA paper folding can be designed to different pattern as required.The paper folding of DNA described in the present invention preferably is two-dimentional DNA paper folding.Described two-dimentional DNA paper folding pattern is preferably the rectangular patterns of 100nm * 70nm.
After wherein the reaction of the described reactant I of step (2) and reactant II reaches balance, just can be imaged on the position that DNA paper folding reactant I modifies by AFM and observe variation highly.The temperature of described molecular balance is preferably 37 ℃.As long as by the number of sites N of the reactant I that designs in the DNA paper folding in the afm image relatively, with the number of sites n of the reactant I that is combined with the reactant II in the afm image, reactant I and reactant II in conjunction with rate when then (n/N) is exactly reaction arrival balance.Therefore, can obtain the reaction equilibrium constant value by the method for counting, the method is the technical method of really on single molecules level biomolecule compatibility feature being studied.
Wherein said atomic force microscope (AFM) formation method is the conventional atomic force microscope formation method in this area.Described atomic force microscope formation method preferably may further comprise the steps: the solution that will react complete drips on mica sheet and adsorbs, the complete mica sheet of absorption is positioned in the liquid tank that is marked with damping fluid, place under the atomic force microscope, carry out the scanning imagery operation in the liquid phase pattern of rapping.
Wherein said adsorption method is the conventional adsorption method in this area.Described adsorption method preferably may further comprise the steps: mica sheet is sticked on the iron plate with double faced adhesive tape, the complete solution example of reaction is dropped on the mica substrate, absorption can place the liquid tank that fills damping fluid after a few minutes, and (AFM) carries out imaging at atomic force microscope.Wherein said mica substrate is the conventional mica substrate in this area, it preferably comprises: with APTES(3-aminocarbonyl propyl-triethoxysilane) mica sheet modified, the mica sheet of modifying with nickel ion or new explanation from mica sheet, the preferred new explanation of the present invention from mica sheet.The time of wherein said absorption is 2～5 minutes preferably, is preferably 5 minutes.The consumption of the solution example that wherein said reaction is complete is that this area AFM detects conventional amount used.The consumption of described sample preferably be＞1 μ L and≤10 μ L, be preferably 2 μ L.The temperature of wherein said AFM imaging is 20～37 ℃ preferably, is preferably 25 ℃.
Wherein said damping fluid is the conventional damping fluid in this area, the preferred 1 * TAE/Mg of the present invention
2+(prescription of described damping fluid is Tris 30～40mM to damping fluid, acetic acid 10～20mM, EDTA 1～2mM, MgCl
210～12.5mM, the pH value is 6～8).The consumption of described damping fluid preferably is 30 μ L～50 μ L, is preferably 50 μ L.Wherein said liquid tank is this area conventional liq groove, and the present invention is preferably provided with the liquid tank in two holes of thin bore conduit.
Wherein said atomic force microscope is the atomic force microscope of this area routine.Described atomic force microscope preferably comprises NANOScope IIIa system or NANOScope VIII type system (Digital Instrument, the U.S.), the preferred NANOScope IIIa of the present invention system.Wherein said afm scan head is this area conventional sweep head, and described afm scan head preferably comprises j-scan head or E type scanner head, the preferred j-scan head of the present invention.Wherein said AFM probe is the conventional AFM probe in this area, and described AFM probe preferably comprises: noncontact/rap pattern probe, contact mode probe, silicon probe or silicon nitride probe.The preferred NP-S10 silicon nitride of AFM probe of the present invention needle point (elasticity coefficient 0.35Nm
-1, bruker company).
Wherein said atomic force microscope imaging pattern is the conventional AFM imaging pattern in this area, and described AFM imaging pattern preferably raps pattern for the AFM liquid phase.Wherein said atomic force microscope imaging makes and firmly is preferably little power imaging.AFM imaging parameters of the present invention is: sweep speed preferably is 1～3Hz, preferred 2Hz; Number of scanning lines preferably is 256～512, preferred 256.
Invent used raw material or reagent except specifying, all commercially available getting.
Than prior art, beneficial effect of the present invention is as follows:
1, biomolecule affinity costant assay method of the present invention in conjunction with the atomic force microscope imaging technique, accurately calculates the method for the affinity costant of biomolecule based on DNA paper folding technology from molecular level.The method has been filled up the blank of measuring biomolecule affinity costant method at single molecules level, for interaction principle and the mechanism of more accurately measuring between the biomolecule provides a kind of method.Simultaneously the present invention also provides the detection means on the microscopic scale for the further research of binding mechanism between the Ag-Ab biomolecule.
2, because the reactant I that the present invention detects and the reaction between the reactant II are specific reactions, therefore method of the present invention has advantages of that specificity is high, simultaneously because DNA paper folding has the advantage that makes the reactant I accurately remain on effective reaction site, therefore the present invention has efficiently the advantage of good reproducibility.In addition, because reaction and imaging only need the microlitre volume, the method also has the few advantage of amount of samples.
Description of drawings
Below in conjunction with description of drawings feature of the present invention and beneficial effect.
Fig. 1 is the schematic diagram in digoxin staple strand site in the embodiment 1DNA paper folding.
Fig. 2 is that embodiment 1 is fixed in the efficiency diagram that the variable concentrations DigiTAb in the DNA paper folding is combined with digoxin; Wherein the final concentration of DNA paper folding is about 1.5nM, the concentration of the DigiTAb among Fig. 2 a is 0.7 μ g/ml, the concentration of the DigiTAb among Fig. 2 b is 0.5 μ g/ml, and the concentration of the DigiTAb among Fig. 2 c is 0.3 μ g/ml, and the concentration of the DigiTAb among Fig. 2 d is 0.2 μ g/ml; Fig. 2 e is the partial enlarged drawing picture of black box diagram picture among Fig. 2 b; Fig. 2 f is the sectional view of embodiment A FM height image: horizontal ordinate is width value (nm of unit), and ordinate is height value (nm of unit).
Fig. 3 is the curve map that embodiment 1 is fixed in the DigiTAb joint efficiency of digoxin in the DNA paper folding and variable concentrations.
Embodiment
The below further specifies the present invention with embodiment, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Framing scaffold chain M13mp18DNA(N4040S) available from NEB company;
DigiTAb is available from Sigma (SIGMA-ALDRICH) company;
The staple strand of the staple strand of unmodified digoxin and modification digoxin is all given birth to worker company available from Shanghai;
Atomic force microscope is NANOScope IIIa system (Digital Instrument, the U.S.), and the AFM scanner head is the j-scan head, and the AFM probe is NP-S10 silicon nitride needle point (elasticity coefficient 0.35Nm
-1, bruker company).
Embodiment 1 measures digoxin and DigiTAb affinity costant
1. modify the preparation of the rectangle DNA paper folding of digoxin
The DNA paper folding of the present embodiment design utilizes M13mp18DNA and the self assembly of staple strand to form the rectangle pattern, and length and width are respectively 100nm and 70nm.As shown in Figure 1, on two positions of DNA paper folding, the end of staple chain is that digoxin is modified, and is used for the combination with DigiTAb.
M13mp18DNA strand and staple strand (comprising the staple strand of modifying digoxin) are mixed by the 1:10 volumetric molar concentration, and place 1 * TAE/Mg
2+Buffer system (Tris 40mM, acetic acid 20mM, EDTA 2mM, MgCl21
2.5mM, the pH value is 8) in, cumulative volume is 61 μ L, place again afterwards on the PCR instrument and be annealed to 20 ℃ with 0.1 ℃/10s of annealing speed from 95 ℃, react complete after, remove unnecessary staple short chain by ultrafiltration, and use the concentration of the described DNA paper folding of determined by ultraviolet spectrophotometry to be 3.0nM, 4 ℃ of preservations got final product after reaction was finished.
2. detect the DigiTAb of variable concentrations and the joint efficiency of the digoxin in the DNA paper folding
Be 4 different concentration with DigiTAb stoste (5.5mg/ml) dilution at first, be respectively 4000 times, 6000 times, 8000 times and 12000 times of dilutions, then respectively with isopyknic DNA paper folding solution hybrid reaction that is connected with digoxin, measure the molecule number that the digoxin of DigiTAb on being fixed on DNA paper folding is combined by the AFM imaging, draw the antigen-antibody binding reaction curve, calculate the affinity costant of biomolecule, concrete assay method may further comprise the steps:
With the DigiTAb of the variable concentrations of 5 μ l respectively with the modification of 5 μ l the DNA paper folding solution of digoxin mix, the DNA paper folding solution of wherein modifying digoxin is step 1 preparation gained, then be put in 37 ℃ the water-bath environment one hour, take out afterwards the complete fluid drips of 2 μ L reaction be added on new explanation from mica on, adsorb after 5 minutes, mica substrate is put into be marked with 50 μ L, 1 * TAE/Mg
2+In the liquid tank in two holes of being furnished with thin bore conduit of damping fluid, rap mode imaging with the AFM liquid phase, image-forming condition is 25 ℃ of temperature, and imaging parameters is sweep speed 2Hz, number of scans 256, and adopt little power imaging.Then collect imaging data, the AFM imaging results is recorded in Fig. 2.
Fig. 2 is digoxin in the DNA paper folding and the afm image of variable concentrations DigiTAb solution reaction, have as seen from the figure that adularescent round point shape thing displays on a plurality of digoxin site (Fig. 2 a～Fig. 2 e), the apparent height amplitude of variation is about 2～3nm(Fig. 2 f), explanatorily be combined with DigiTAb in the Gaoxin.The white bright spot number of AFM detection gained is the complex molecule number of digoxin and DigiTAb.
Count the N that adds up to of scanning area scope Endoxin antigen molecule number according to the AFM imaging data, the digoxin antigen molecule number of being combined with antibody is n, then (n/N) be digoxin-DigiTAb in conjunction with rate.
According to the data analysis of above step gained differently the Gaoxin antibody concentration in conjunction with rate, its result is respectively: be 95.5% in conjunction with rate when DigiTAb concentration is 0.7 μ g/ml; Be 59.1% in conjunction with rate when DigiTAb concentration is 0.5 μ g/ml; Be 43.7% in conjunction with rate when DigiTAb concentration is 0.3 μ g/ml; Be 23.5% in conjunction with rate when DigiTAb concentration is 0.2 μ g/ml.According to above data, take antibody concentration as X-axis, the percentage that the conjugated antigen molecule accounts for total antigen molecule is Y-axis, draws the antigen-antibody binding reaction curve and carries out as a result match, and its result as shown in Figure 3.
3. calculate digoxin-DigiTAb affinity costant
Calculate affinity costant take digoxin-DigiTAb as 50% the time in conjunction with rate, as can be known from Fig. 1,2 digoxin (DIG) design site is arranged in every DNA paper folding, each antigen can be in conjunction with 1 DigiTAb.The concentration of DNA paper folding can be measured wavelength by ultraviolet-visible pectrophotometer and measure at the absorption peak of 260nm chamber, is 3.0nM through calculating its DNA paper folding concentration value.With this solution and equal-volume antibody-solutions phase hybrid reaction.Among Fig. 2, single antigen and antibody complex number can clearly observe, and free antigen and can obtain in conjunction with the rate situation of change concerns one to one according to antigen and antibody response again, also can obtain free antibodies concentration.Can see from the curve of Fig. 3: when being 50% in conjunction with rate, namely the final concentration of antibody is 2.4 * 10
-9(the antibody initial concentration is 5.5mg/ml, and the molecular weight of establishing antibody is 160000Da, and then its volumetric molar concentration is 5.5/160000=0.34mM during M.Diluted 7100 times this moment, and then its volumetric molar concentration is 0.34/7100=4.8nM).
According to following formula: K=[AbAg]/[Ag] [Ab], nM=1.1 * 10 of its K=0.75nM/0.75nM * (2.4-1.5)
-9M, its as a result K be the digoxin calculated by the method for the invention and the affinity costant of DigiTAb.Occur although the mica outside paper folding is understood some other bright spot, the statistics that does not affect the bright spot on our folio affects.
Than existing biomolecule affinity costant assay method such as enzyme-linked immunosorbent assay (Conan, (1985) ELISA-based determination of immunological binding constants.Mol.Immunol.22.321.) and surface plasma body resonant vibration method (Laurence Heinrich K.N.L., Nathalie Tissot, Daniel Jean et.al, Comparison of the results obtained by ELISA and surface plasmon resonance for the determination of antibody affinity, Journal of Immunological Methods 352 (2010) 13 – 22), the biomolecule affinity costant assay method based on DNA paper folding of the present invention has efficiently, accurate rate is high, the advantage that good reproducibility and amount of samples are few.
Therefore, the biomolecule affinity costant mensuration based on DNA paper folding provided by the invention can reach direct from the interaction mechanism between the Molecular level study biological immune molecule, and this has very important impetus for immunologic development; Simultaneously the present invention also provides a kind of new research method and means for the interaction mechanism between the other biological molecule.
Should be understood that after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. the affinity costant assay method based on the biomolecule of DNA paper folding is characterized in that, described biomolecule comprises reactant I and the reactant II that can be bonded to each other and dissociate, and this assay method may further comprise the steps:
(1) the reactant I is connected in DNA paper folding staple chain end, then forms DNA paper folding with the self assembly of framing scaffold chain;
(2) add the reactant II, after reaching balance, reaction utilizes the afm scan imaging collection data, calculate the concentration [AbAg] of reactant I and reactant II compound, the concentration [Ab] of the concentration [Ag] of free reactant I and free reactant II;
(3) with the following formula of step (2) institute's value substitution, K=[AbAg]/[Ag] [Ab], calculate the affinity costant K of described reactant I and reactant II.
2. assay method as claimed in claim 1 is characterized in that, described reactant I and reactant II comprise: antigen and antibody, biotin and Avidin, in aptamer and target molecule thereof and part and the acceptor one group or several groups.
3. assay method as claimed in claim 2 is characterized in that, described reactant I and reactant II are digoxin and DigiTAb.
4. assay method as claimed in claim 1 is characterized in that, the described staple chain of step (1) and the self assembly of framing scaffold chain form DNA paper folding and comprise: the framing scaffold chain is mixed being placed on 1 * TAE/Mg with the staple chain
2+Buffer solution system, the end of wherein said staple chain is connected with the reactant I, and the mol ratio of described framing scaffold chain and staple chain is 1:10～1:5, places on the PCR instrument again and is annealed to 20 ℃ from 95 ℃, annealing speed is 0.1 ℃/10 seconds, namely forms described DNA paper folding.
5. assay method as claimed in claim 4 is characterized in that, the DNA paper folding that described staple chain and the self assembly of framing scaffold chain form is two-dimentional DNA paper folding.
6. assay method as claimed in claim 5 is characterized in that, the rectangle DNA paper folding that described two-dimentional DNA paper folding is 100nm * 70nm.
7. assay method as claimed in claim 1, it is characterized in that, the described atomic force microscope imaging method of step (2) may further comprise the steps: the solution that reaction is reached balance drips on mica sheet and adsorbs, the complete mica sheet of absorption is positioned in the liquid tank that is marked with damping fluid, place under the atomic force microscope, carry out the scanning imagery operation in the liquid phase pattern of rapping.
8. assay method as claimed in claim 7 is characterized in that, described mica sheet be new explanation from mica sheet, the time of described absorption is 2～5 minutes.
9. assay method as claimed in claim 7 is characterized in that, described damping fluid is 1 * TAE/Mg
2+Damping fluid, the consumption of described damping fluid are 30 μ L～50 μ L, and described liquid tank is the liquid tank of being furnished with two holes of thin bore conduit.
10. assay method as claimed in claim 7 is characterized in that, described atomic force microscope imaging method is: use little power imaging; The speed of described scanning is 1～3Hz, and the line number of described scanning is 256～512.
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| CN107469088A (en) * | 2017-06-27 | 2017-12-15 | 郑州大学 | A kind of construction method of accurate identification targeted nano carrier based on DNA paper folding arts and its application |
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