CN106757377B - A kind of aptamers combinatorial libraries, its construction method and purposes - Google Patents

Abstract

The present invention provides a kind of method of building aptamers combinatorial libraries, the aptamers combinatorial libraries that the method based on above-mentioned building aptamers combinatorial libraries constructs, the method for aptamers is screened by constructed aptamers combinatorial libraries and screens the aptamers of acquisition by above-mentioned screening technique.The method of building aptamers combinatorial libraries provided by the invention, the combinatorial libraries for including less sequence is constructed using efficient structural tailoring mode, constructed aptamers combinatorial libraries cover the most of possible ranges that can generate special affinity interaction.The present invention screens to obtain the most short aptamers bioactive sequence of more excellent, the irredundant sequence of affinity.

Description

A kind of aptamers combinatorial libraries, its construction method and purposes

Technical field

The present invention relates to molecular biology, analytical chemistry and combinatorial chemistry fields.Specifically, the present invention relates to adaptations Body combinatorial libraries and its construction method further relate to the method using aptamers needed for the aptamers combinatorial libraries screening and assessment And the aptamers screened by this method.The invention further relates to the purposes of the aptamers screened and obtained and containing described Screen the kit of obtained aptamers.It is screened using aptamers combinatorial libraries provided by the invention and screening technique suitable Ligand affinity is more excellent, and irredundant sequence.

Background technique

Oligonucleotides aptamers (Oligonucleotide aptamer), usually the aglucon phyletic evolution through index concentration Technology (Systematic Evolution of Ligands by Exponential Enrichment, SELEX) screening obtains (Nature 1990,346；Science 1990,249 (4968): 505-510), generally one section short single-stranded oligonucleotide sequence It arranges (ssDNA or RNA), by being folded into certain three-dimensional structure, forms high-affinity, height through steric configuration complementation and target molecule Specific binding, also known as " chemical antibody " (Oncotarget 2016,7 (12): 13446-13463).

Overall length aptamers are also known as the aptamers obtained by SELEX technology screening, generally comprising primer sequence and Random sequence, length is generally between 70~130 bases (nt).The key binding sites of overall length aptamers and target molecule are generally simultaneously Indefinite, which results in overall length aptamers to be not suitable as functional recognition component.Many adaptations gone out through SELEX technology screening Body, guiding region and random area generate base pairing, and certain guiding regions take part in specific recognition, or random region sequence compared with Long, there are redundancy base sequences.Therefore, for overall length aptamers sequence, structural tailoring need to be carried out to it, in order to both be protected It holds in conjunction with activity, only the aptamers of suitable length (10-40nt), such aptamers are more suitable for functional identification member again Part.Such aptamers in order to obtain need accurately to define the aptamers sequence (or structural domain) that contribution is generated to identification function.

Therefore, (post-SELEX) is screened after aptamers is one of research hotspot always, and structural tailoring, chemistry has been developed The aptamers that compatibility is higher, specificity is stronger, sequence is shorter are therefrom found in the methods of modification, multivalent combination, rite-directed mutagenesis, But majority is still limited to be attempted with trial-and-error method (J.Biol.Chem.2001,276 (54): 48644-48654).

Enriched sequence of the trial-and-error method generally be directed to end wheel library, the lower freedom obtained by shared secondary structure and calculating On the basis of energy (Δ G), according to the empirical judgement of some nucleic acid secondary structures, more random cut is carried out to aptamers sequence Sanction or rite-directed mutagenesis, for example, upper official rank uses trial-and-error method, what is screened to it is in 3 second level knot of ring 1- stem 1- ring 2- stem 2- ring The lung carcinoma cell aptamers Sgc8 of structure carries out structural tailoring, cuts out ring 1, stem 1, ring 2 etc. step by step, finally obtains the suitable of stem 2- ring 3 Ligand sequence Sgc8c remains the affinity (Chem.Bio.Chem.2007,8:603-606) of former Sgc8 substantially.In another example Inventor is directed to the overall length aptamers 828 that recombinant human erythropoietin-α is screened before, is cut out whole stochastic orderings The 828r obtained after column, the slightly excellent (CN102191250A of more originally 828 affinity；Bioorg.Med.Chem.2010,18, 8016-8025)。

Above-mentioned trial-and-error method efficiency is lower, and often screening obtains many invalid sequences.For example, Ferreira-Bravo etc. is used Trial-and-error method carries out base replacement to the DNA aptamers FA1 of the HIV reverse transcription albumen obtained through SELEX technology screening, only will adaptation Four stem's complementary base sequence whole simple replacements of body are G-C base-pair, as a result, it has been found that the replacement is to affine activity without bright Development is rung；Simultaneously for four ring portion structural domains of aptamers, only carries out simple randomization and cut out, also do not find that affine activity is excellent Punctured sequence (Nucleic Acids Res.2015,43 (20): 9587-9599).In another example Esposito etc. is directed to 15nt long Thrombin aptamer TBA, create 14 in the reversion of different loci polarity (from 5 ' -3 ' overturnings for 3 ' -5 ') sequence, though can be into Row structure elucidation, but not active more preferably aptamers (the Nucleosides Nucleotides Nucleic Acids of discovery 2007,26(8-9):1145-1149).The above method is difficult in limited sequence context, and it is more excellent, special to obtain affine activity Stronger optimization aptamers (Improved aptamer) sequence of property.

There are also the scholars such as Nonaka to introduce the field post-SELEX for the virtual screening based on genetic algorithm, assigns next-generation The higher occurrence probability of high-affinity sequence in library, combining random mutational site are sieved in a plurality of isometric mutant nucleotide sequence of 3 generation 50 It chooses compared with high 10 times of the aptamers (Anal.Chem.2013,85,1132-1137) of the compatibility of former VEGF aptamers sequence. But still the problem whether aptamers sequence is guarded can not be answered, it can not also determine that those are critical bases.Illustrate currently in post- The method that trial-and-error method generally continued to use in SELEX etc. carries out structural tailoring or rite-directed mutagenesis, working efficiency is lower, difficulty is larger, Still planless theoretical direction and preferable solution.

When screening and optimizing aptamers, need to characterize and evaluate affinity between aptamers and target molecule.Currently, being used for table It seeks peace the method for evaluating affinity between aptamers and target molecule, mainly includes surface plasmon resonance (Surface Plasmon Resonance, SPR), biomembrane interferometry (Bio-Layer Interferometry, BLI), isothermal titration amount Thermal method (Isothermal Calorimetry, ITC), gel electrophoresis mobility variation analysis method (Electrophoretic Mobility Shift Assay, EMSA), micro thermophoresis moves method (Microscale Thermophoresis, MST), backward dissipates Penetrate interferometry (Backscattering Interferometry, BSI) and Media by Fluorescence Anisotropy (Fluorescence Polarization, FP) etc., change according to the characteristic value before and after combination to determine affinity between aptamers and target molecule. EMSA, FP, MST are both needed to carry out fluorescence or radioactive label to target molecule or ligand；ITC belongs to label-free analytical technology, but spirit Sensitivity is lower, sample size need to be used larger；SPR, BLI, BSI and MST based on molecule autofluorescence etc. are then because its is label-free, sensitive Spend high be concerned.SPR and BLI can also provide dynamics data other than being capable of providing affinity data simultaneously, can be real Existing high throughput assay.SPR and BLI is both needed to that target molecule is fixed, by target molecule and solution of the reading surface after fixed Ligand between act on after the variation of surface plasma body resonant vibration signal strength or the increase of surface thickness and implement to measure.

When using SPR technique, after needing the fixed form to target molecule, the solution system of interaction, interaction The key factors such as regeneration condition investigated and optimized, can after establishing suitable, stable, highly sensitive SPR method system Carry out the work such as subsequent aptamers sequence screening evaluation.

In order to screen, optimize the most short aptamers bioactive sequence of more excellent, the irredundant sequence of affinity, need to look for and open Send out method of cutting out suitable and structural tailoring carried out to overall length aptamers (or aptamers sequence to be optimized), it is also necessary to establish it is suitable, Stable, highly sensitive characterization and evaluation method, so as to efficiently evaluate the affinity between institute punctured sequence and target molecule.

Summary of the invention

It is an object of the present invention to provide a kind of methods for constructing aptamers combinatorial libraries.

The method building based on above-mentioned building aptamers combinatorial libraries that it is a further object to provide a kind of is fitted Ligand combinatorial library.

A further object of the present invention is to provide constructed by a kind of method by above-mentioned building aptamers combinatorial libraries The method of aptamers combinatorial libraries screening aptamers.

A further object of the present invention is to provide the aptamers screened by above-mentioned screening technique.

A further object of the present invention is to provide the purposes for the aptamers screened by above-mentioned screening technique.

It is also another object of the present invention to provide the kits containing the aptamers screened by above-mentioned screening technique.

It is also another object of the present invention to provide the compositions containing the aptamers screened by above-mentioned screening technique.

It is also another object of the present invention to provide the chips containing the aptamers screened by above-mentioned screening technique.

To achieve the above object, the present invention provides a kind of method for constructing aptamers combinatorial libraries, comprising the following steps:

The aptamers to be optimized with N number of base are provided, using aptamers to be optimized as initiation sequence；

The first molecular group is constructed, it includes n₁A molecule, the n₁5 ' the ends that a molecule is respectively initiation sequence truncate Body, and each molecule relative to initiation sequence there are 5 ' different ends to truncate length；

The second molecular group is constructed, it includes n₂A molecule, the n₂3 ' the ends that a molecule is respectively initiation sequence truncate Body, and each molecule relative to initiation sequence there are 3 ' different ends to truncate length；

Third molecular group is constructed, it includes n₃A molecule, the n₃5 ' and 3 ' ends that a molecule is respectively initiation sequence are same When truncated truncate, 5 ' with 3 ' ends to truncate length identical in a molecule, and each molecule is relative to initial sequence Arranging, there is different ends to truncate length；

Optionally, the 4th molecular group is constructed, it includes n₄A molecule,

When initiation sequence is capable of forming loop-stem structure, the n₄A molecule is respectively the ring portion Series extension of initiation sequence Body, the ring portion Series extension body do not include stem's sequence of initiation sequence, include 1~n in 5 ' ends₄A First ray (example The sequence being such as made of A and G, such as AG), and include 1~n in 3 ' ends₄A second sequence, second sequence are the first sequences The reverse complementary sequence (such as the sequence being made of C and T, such as CT) of column, the first sequence that 5 ' ends include in a molecule Column are identical with the second sequence quantity that 3 ' ends include, and each molecule relative to initiation sequence there is different ends to prolong Length；

When initiation sequence cannot form loop-stem structure, the n₄A molecule is respectively the extension body of initiation sequence, described 5 ' ends of extension body include 1~n₄A First ray (such as the sequence being made of A and G, such as AG), and include in 3 ' ends 1~n₄A second sequence, second sequence are reverse complementary sequence (such as the sequence being made of C and T, the example of First ray Such as CT), the second sequence quantity that the First ray and 3 ' ends that 5 ' ends include in a molecule include is identical, and every One molecule has different terminal extension length relative to initiation sequence,

The length of the First ray can be one or more bases, for example, 1,2,3 or 4 base；

Wherein, N is the integer (for example, integer between 30~130) greater than zero,

n₁、n₂、n₃、n₄The integer greater than zero and less than or equal to N is each independently (for example, whole between 5~80 Number).

In another preferred embodiment, the method for building aptamers combinatorial libraries of the present invention, wherein

The construction method of first molecular group are as follows: initiation sequence is truncated from 5 ' ends, carries out n altogether₁It is secondary, it truncates every time One or more (for example, 1,2,3,4) bases, thus to obtain n₁A truncated molecule in 5 ' end constitutes the first molecular group；

The construction method of second molecular group are as follows: initiation sequence is truncated from 3 ' ends, carries out n altogether₂It is secondary, it truncates every time One or more (for example, 1,2,3,4) bases, thus to obtain n₂A truncated molecule in 3 ' end constitutes the second molecular group；

The construction method of third molecular group are as follows: initiation sequence is truncated simultaneously from 5 ' and 3 ' ends, carries out n altogether₃It is secondary, One or more (for example, 1,2,3,4) bases are truncated simultaneously to 5 ' and 3 ' ends every time, thus to obtain n₃A 5 ' and 3 ' ends Truncated molecule simultaneously constitutes third molecular group；

The construction method of 4th molecular group are as follows:

When initiation sequence is capable of forming loop-stem structure, keeps the ring portion sequence of initiation sequence constant, stem's sequence is gone It removes, in 5 ' ends of ring portion sequence addition First ray (such as the sequence being made of A and G, such as AG), and in ring portion sequence The second sequence is added in 3 ' ends, second sequence be First ray reverse complementary sequence (such as the sequence being made of C and T, Such as CT), repeat n₄It is secondary, thus to obtain n₄A 5 ' and 3 ' ends while extended molecule constitute the 4th molecular group；

When initiation sequence cannot form loop-stem structure, 5 ' ends of initiation sequence addition First ray (such as by A and The sequence of G composition, such as AG), and the second sequence is added in 3 ' ends, second sequence is the reverse complemental sequence of First ray Column (such as the sequence being made of C and T, such as CT), repeat n₄It is secondary, thus to obtain n₄A 5 ' and 3 ' ends are extended simultaneously Molecule constitutes the 4th molecular group.

In another preferred embodiment, the method for building aptamers combinatorial libraries of the present invention, wherein the The shortest molecule of length in one molecular group, the second molecular group or third molecular group is with 10~40 bases, for example, 20 ~30 bases.

In another preferred embodiment, the method for building aptamers combinatorial libraries of the present invention, wherein institute Stating aptamers to be optimized is to specifically bind the aptamers of epo protein, for example, overall length aptamers shown in SEQ ID NO.1.

In another preferred embodiment, the method for building aptamers combinatorial libraries of the present invention, wherein institute The aptamers that aptamers to be optimized are specific binding epo protein are stated, for example, overall length aptamers shown in SEQ ID NO.1, Middle N=39, n₁=9, n₂=9, n₃=9, n₄=8, the length of the First ray is 1 base, 2 bases or 3 bases.

The present invention also provides a kind of aptamers combinatorial libraries, comprising:

First molecular group, it includes n₁A molecule, the n₁A molecule is respectively 5 ' end truncates of initiation sequence, and And each molecule relative to initiation sequence there are 5 ' different ends to truncate length；

Second molecular group, it includes n₂A molecule, the n₂A molecule is respectively 3 ' end truncates of initiation sequence, and And each molecule relative to initiation sequence there are 3 ' different ends to truncate length；

Third molecular group, it includes n₃A molecule, the n₃A molecule is respectively 5 ' and 3 ' ends of initiation sequence while cutting Short truncate, 5 ' is identical with 3 ' ends truncation length in a molecule, and each molecule has relative to initiation sequence There is different ends to truncate length；And

Optionally, the 4th molecular group, it includes n₄A molecule,

When initiation sequence is capable of forming loop-stem structure, the n₄A molecule is respectively the ring portion Series extension of initiation sequence Body, the ring portion Series extension body do not include stem's sequence of initiation sequence, include 1~n in 5 ' ends₄A First ray (example The sequence being such as made of A and G, such as AG), and include 1~n in 3 ' ends₄A second sequence, second sequence are the first sequences The reverse complementary sequence (such as the sequence being made of C and T, such as CT) of column, the first sequence that 5 ' ends include in a molecule Column are identical with the second sequence quantity that 3 ' ends include, and each molecule relative to initiation sequence there is different ends to prolong Length；

When initiation sequence cannot form loop-stem structure, the n₄A molecule is respectively the extension body of initiation sequence, described 5 ' ends of extension body include 1~n₄A First ray (such as the sequence being made of A and G, such as AG), and include in 3 ' ends 1~n₄A second sequence, second sequence are reverse complementary sequence (such as the sequence being made of C and T, the example of First ray Such as CT), the second sequence quantity that the First ray and 3 ' ends that 5 ' ends include in a molecule include is identical and each A molecule has different terminal extension length relative to initiation sequence,

The length of the First ray can be one or more bases, for example, 1,2,3,4 base；

Wherein, N is the integer (for example, integer between 30~130) greater than zero,

n₁、n₂、n₃、n₄The integer greater than zero and less than or equal to N is each independently (for example, whole between 5~80 Number),

The initiation sequence is aptamers to be optimized.

In a preferred embodiment, aptamers combinatorial libraries of the present invention, wherein the adaptation to be optimized Body is the aptamers for specifically binding epo protein, for example, overall length aptamers shown in SEQ ID NO.1.

In another preferred embodiment, aptamers combinatorial libraries of the present invention, by SEQ ID NO.2~ Sequence composition shown in SEQ ID NO.36.

The present invention also provides a kind of method for screening aptamers, this method uses adaptation described in aforementioned Arbitrary Term of the present invention Aptamers needed for body combinatorial libraries screen, for example, the aptamers of epo protein can be specifically bound.

In a preferred embodiment, the screening technique of aptamers of the present invention comprising:

(1) aptamers combinatorial libraries are provided；

(2) each of aptamers combinatorial libraries molecule is contacted with target protein (such as epo protein)；

(3) affinity in aptamers combinatorial libraries between each molecule and target protein is detected.

In a preferred embodiment, the screening technique of aptamers of the present invention, this method is using surface etc. Gas ions resonance method (SPR method) detects the affinity between each molecule and target protein.

In another preferred embodiment, aptamers screening technique of the present invention, comprising the following steps:

1) target protein is fixed on chip (such as SA chip) surface；

2) SPR dynamic analysis is carried out, sample introduction makes the molecule in the aptamers combinatorial libraries and is fixed on chip surface Target protein combine, generate compound, sample introduction terminates, and injects blank buffer solution, and the compound on chip is dissociated, obtained Each sequence single point R U value change curve is worth (RU value) judges whether aptamers to be screened are active according to response, and RU value is greater than 0 Aptamers it is active；

3) it for active molecule, carries out multi-cycle dynamic analysis (MCK analysis), obtains dissociation constant K_D, root According to K_DThe size of numerical value judges the affinity between each molecule and target protein, K_DNumerical value is smaller, and affinity is stronger.

In another preferred embodiment, the screening technique of aptamers of the present invention or any applicable above Embodiment, wherein step 1) is will to be fixed on chip surface after the target protein couple biotin, for example, the target protein In the sialic acid terminal and biotin covalent coupling of the amino terminal of amino acid residue, carboxyl terminal or glycosyl, preferably target protein Stoichiometric ratio with biotin covalent coupling is 1:1.

In another preferred embodiment, the screening technique of aptamers of the present invention or any applicable above Embodiment, wherein molecule in step 2) the aptamers combinatorial libraries is denaturalized 5 in 90~98 DEG C (such as 95 DEG C)~ 10min (such as 5min), cooled to room temperature, then sample introduction, injector temperature is 20~30 DEG C (such as 25 DEG C), when sample introduction combines Between be 60~180 seconds (such as 120 seconds), Dissociation time be 120~600 seconds (such as 300 seconds), flow velocity be 20~50 μ L/min (such as 30 μ L/min), buffer used are Tris-T buffer.

In another preferred embodiment, the screening technique of aptamers of the present invention or any applicable above Embodiment, aptamers screening technique of the present invention, wherein the aptamers are combined in the analysis of MCK described in step 3) Molecule in library in 90~98 DEG C (such as 95 DEG C) denaturation 5~10min (such as 5min), cooled to room temperature, then sample introduction, Injector temperature is 20~30 DEG C (such as 25 DEG C), and sample introduction binding time is 60~180 seconds (such as 120 seconds), Dissociation time 120 ~600 seconds (such as 300 seconds), flow velocity was 20~50 μ L/min (such as 30 μ L/min), and buffer used is in MCK analytic process Tris-T buffer.

In another preferred embodiment, the screening technique of aptamers of the present invention or any applicable above Embodiment, wherein the formula of the Tris-T buffer are as follows: 20mM Tris, 140mM NaCl, 5mM MgCl2,5mM KCl, Tween 20 is adjusted to pH with HCl as 7.4-7.6.Wherein, 20 concentration range of Tween is 0.005%~0.1%, such as It is 0.05%.

In another preferred embodiment, the screening technique of aptamers of the present invention or any applicable above Embodiment, wherein the regeneration condition of chip can be the salt of high ionic strength, such as 1~3M NaCl, such as 3M NaCl； It may be acid condition, such as the 10mM glycine that pH is 1.5~2.5, such as the 10mM glycine that pH is 2.5；It can be with For alkaline condition, such as 1-15mM NaOH, such as 5mM NaOH.Preferred regeneration condition is 5mM NaOH, reproduction time 15 Second.

The present invention also provides the aptamers that one kind can specifically bind epo protein, are selected from SEQ ID NO.4~9 institute The sequence shown, and the sequence as shown in Formulas I to Formula VII,

N-AGGT-Y-TGTTTTTGGGGTTGGTTTGGG (Formulas I)

AGAGAGAGAGAGAGA-N-AGGT-Y-TGTTTTTGGGGTTGG-TTTGGG-TCTCTC TCTCTCTCT (formula II)

GAGAGAGAGAGA-N-AGGT-Y-TGTTTTTGGGGTTGGTTT-GGG-TCTCTCTCTCT C (formula III)

GAGAGAGAGA-N-AGGT-Y-TGTTTTTGGGGTTGGTTT-GGG-TCTCTCTCTC (formula IV)

AGAGAGA-N-AGGT-Y-TGTTTTTGGGGTTGGTTTGGG-TCTCTCT (Formula V)

GAGAGA-N-AGGT-Y-TGTTTTTGGGGTTGGTTTGGG-TCTCTC (Formula IV)

AGAGA-N-AGGT-Y-TGTTTTTGGGGTTGGTTTGGG-TCTCT (Formula VII)

Wherein, N G, A, C or T；Y is C or T.

The present invention also provides the purposes that the aptamers that can specifically bind epo protein are used to detect epo protein.

The present invention also provides the compositions, kit or chip for detecting epo protein, wherein containing aforementioned present invention institute That states can specifically bind the aptamers of epo protein.

The present invention also provides a kind of methods for detecting epo protein, comprising:

(1) sample to be tested is provided；

(2) aptamers described in claim 16 are contacted with sample to be tested；

(3) combination between aptamers and EPO is detected.

In the present invention, the aptamers to be optimized can be any and target molecule and form high-affinity or high specific In conjunction with aptamers, such as the aptamers of epo protein can be specifically bound.

In the present invention, the epo protein can be natural EPO albumen, rHuEPO- α albumen or rHuEP O- β albumen, It is also possible to deglycosylated above-mentioned epo protein.

Detailed description of the invention

Inventor's selection recombinant human erythropoietin-α (recombinant human Erythropoietin- α, RHuEPO- α, abbreviation EPO- α) single stranded DNA aptamers 828r as initiation sequence or model molecule (referring to CN102191250A；Bioorg.Med.Chem.2010,18,8016-8025), illustrate building adaptation of the invention The method of body combinatorial libraries, the aptamers combinatorial libraries of the method building based on above-mentioned building aptamers combinatorial libraries, Yi Jitong The method for crossing aptamers needed for constructed aptamers combinatorial libraries screen.

The single stranded DNA aptamers of the EPO- α as shown in SEQ ID NO:1, sequence length 39nt, in the present invention It is named as 828r-39nt (abbreviation 39nt).The secondary structure of 39nt is in the dissociation constant K between stem ring shape, with EPO- α_D K is measured as by EMSA method_{D(39nt/EPO-α)}Specific action site between=39 ± 27nM, with EPO- α is still not clear.

Inventor then dexterously by means of combinatorial chemistry thinking, proposes that " adjacent base site most probably passes through alkali in sequence The hypothesis of base accumulation influence steric interaction ", invention devise four groups of stepping type sequences, constitute stepping type aptamers combination text Library.Four groups of sequences in the library are respectively designated as shrinking (Left contraction) molecular group to the left, shrink to the right (Right contraction) molecular group, both ends shrink (Inner contraction) molecular group and both ends extend (also known as stem Portion extends, Stem-nbp) molecular group.

Wherein, molecular group is shunk to the left to refer on the basis of initiation sequence 39nt, puncture phase step by step from its 5 ' end Two adjacent bases, constitute from Left contraction 37,35,33 ..., until Left contraction 21 (abbreviation L37, L35, L33 ..., L21) etc. 9 sequences.It is then exactly the opposite that molecular group is shunk to the right, is in initiation sequence 807- On the basis of 39nt, puncture two adjacent bases step by step from its 3 ' end, constitute from Right contraction 37, 35,33 ..., until Right contraction 21 (abbreviation R37, R35, R33 ..., R21) etc. 9 sequences.Both ends It shrinks molecular group and refers to that on the basis of initiation sequence 39nt, two sides respectively puncture single base step by step from its 5 ' and 3 ' end, Constitute from Inner contraction 37,35,33 ..., until Inner contraction 21 (abbreviation In37, In35, In33 ..., In21) etc. 9 sequences.Such three groups each 9 sequences, are obtained 27 sequences (referring to Fig. 1).Stem Extend molecular group then to refer in the case where keeping aptamers ring portion sequence constant, stem's sequence is changed to First ray and (such as wraps Sequence containing G and A, for example, GA, AGA, AGAGA, GAGAGA, AGAGAGA, GAGAGAGAGA, GAGAGAGAGAGA, AGAGAGAGAGAGAGA) ... the second sequence (such as the sequence comprising C and T, for example, TC, TCT, TCTCT, TCTCTC, TCTCTCT, TCTCTCTCTC, TCTCTCTCTCTC, TCTCTCTCTCTCTCT) complementary base, constitute from Stem-2bp, 3bp, 8 sequences such as 5bp, 6bp, 7bp, 10bp, 12bp to Stem-15bp.Thus configured aptamers stepping type combinatorial libraries, are only used 35 sequences cover the most of possible ranges that can generate special affinity interaction, while can define specificity adaptation The effect in critical base site in body.This is just to obtain most short aptamers, and inquire into possible binding mechanism and lay a good foundation.

The present inventor screens the high specificity of affine activity using above-mentioned constructed stepping type aptamers combinatorial libraries and ties Close the aptamers of EPO- α albumen.When screening, affinity between aptamers and target molecule is characterized and evaluated using SPR method.Specifically, Used instrument can be Biacore T100, T200 (GE company, the U.S.), ProteOn XPR36 (Bio Rad Laboratories), Reichert4SPR (Reichert company, the U.S.) etc..

When measuring affine activity, using the coated Dextran Chip of Streptavidin (abbreviation SA chip) fixed target protein EPO-α.After EPO- α and biotin covalent coupling, it is fixed on SA chip surface.

EPO- α is known as biotinylation EPO- α with the product after biotin covalent coupling.EPO- α can be in several ways With biotin covalent coupling, the mode of covalent coupling depends on the end group type in EPO- alpha molecule structure.For example, described EPO- α can be coupled by the terminal amino group couple biotin of its amino acid residue or the terminal carboxyl group of its amino acid residue Biotin, or the terminal sialic acid groups couple biotin of its glycosyl can be passed through.

Biotin group, reactive group and linking arm part are generally divided into the structure of coupling reagent.Connection appropriate Steric hindrance when arm lengths effectively can overcome fixed.Different couplings can be selected according to the type of EPO- α end group Reagent.

For example, the biotin coupling reagent selected can be for not when the amino acid residue end of EPO- α is amino group With the polysuccinimated biotin of form, such as Sulfo-NHS-LC-Biotin (Sulfosuccinimidyl-6- [biotin-amido] hexanoate, 6- [biotin amido group] caproic acid sulfonic group-N-hydroxy-succinamide ester), Sulfo- NHS-LC-LC-Biotin (Sulfosuccinimidyl-6- [biotin-amido] -6-hexanamido hexanoate, 6- [biotin amido group] -6- hexanoyl aminocaproic acid-sulfonic group-N-hydroxy-succinamide ester), NHS-LC-Biotin (Succinimidyl 6- (biotinamido) hexanoate, 6- [biotin amido group] caproic acid-n-hydroxysuccinimide Ester), NHS-LC-LC-Biotin (Succinimidyl-6- (biotin-amido) -6-hexanamido hexanoate, 6- [biotin amido group] -6- hexanoyl aminocaproic acid-N-hydroxy-succinamide ester), (biotin four is poly- by NHS-PEG4-Biotin Ethylene glycol N-hydroxy-succinamide ester), NHS-PEG12-Biotin (12 polyethylene glycol n-hydroxysuccinimide of biotin Ester) etc..

When the amino acid residue end of EPO- α is carboxylic group, the biotin coupling reagent selected can be end ammonia Base biotin, for example, Amine-PEG3-Biotin (three polyoxamide of biotin), Biocytin Hydrazide (biology Plain hydrazides) etc., carbodiimide (EDC) need to be added in when coupling simultaneously.

When the glycosyl end of EPO- α is sialic acid, the biotin coupling reagent selected can be biotinylated hydrazine, example Such as Biocytin Hydrazide (biotin hydrazides), Biotin-LC-Hydrazide (6- (biotinamido) Hexanehydrazide, 6- [biotin amide]-hexanoyl hydrazine), Biotin-PEG4-Hydrazide (four polyethylene glycol of biotin Hydrazides), need when coupling first by sialic acids groups with sodium periodate oxidation at aldehyde.

In a preferred embodiment, EPO- α and coupling reagent are with 1:1 molar ratio covalent coupling, referred to as unimolecule Biotinylation EPO- α albumen, abbreviation Bio-EPO- α.

In a preferred embodiment, when the amino acid residue end of EPO- α is amino group, coupling reagent is chosen Sulfo-NHS-LC-Biotin and EPO- α covalent coupling, obtain the biotinylated NH of unimolecule₂-Bio-EPO-α.EPO- α's When amino acid residue end is carboxylic group, coupling reagent Biotin Hydrazide and EPO- α covalent coupling are chosen, list is obtained The COOH-Bio-EPO- α of molecular biosciences element.When the glycosyl end of EPO- α is sialic acids groups, coupling reagent is chosen Biocytin Hydrazide and EPO- α covalent coupling, obtain the biotinylated Sialyl-Bio-EPO- α of unimolecule.

In the fixed target protein of chip surface, according to different experiment purposes, the target protein that can choose different level is dense Degree.When carrying out multi-cycle dynamics (MCK) analysis, suitable concentration is selected, the Bmax (Rmax) of target analytes is made ≤ 30 response units (RU).And if carry out steady-state analysis, target protein concentration can be higher, so that fixed amount reaches full With.Ratio is measured in Rmax=ligand fixed amount × (molecular weight/ligand molecular weight of target analytes) × combination.

In the fixed target protein of chip surface, it is also necessary to investigate and optimize the conditions such as set time, flow velocity.Of the invention In one embodiment, channel initialization is carried out with 50mM NaOH, 1M NaCl solution first, sample introduction 3 times, flow velocity is 20 μ L/ Min, the time of each sample introduction are 60 seconds, and then chip is normalized with 70% glycerine water solution.Then by 1 μM Unimolecule biotinylated Sialyl-Bio-EPO or NH₂- Bio-EPO- α, in 10 μ L/min of flow velocity, sample injection time 60 seconds Under the conditions of, it is fixed on SA chip.Regeneration condition is 5mM NaOH, and 30 μ L/min of flow velocity, the reproduction time is 15 seconds.

In a preferred embodiment, the present invention carries out SPR dynamic analysis experiment using single-point input mode, i.e., Single-point screening method filters out the sequence (also known as bioactive sequence) in each adaptor molecules group with EPO- α with affinity, specific table Now there is positive response for combination-dissociation curve (i.e. after sample introduction, the change curve of response (RU value)).Single-point sample introduction, that is, Single concentration sample introduction, can intuitively represent whether sample exists with target protein in conjunction with active response (i.e. value > 0 RU)).

SPR dynamic analysis can determine in a given time span, aptamers and the target being fixed on chip Whether the compound that albumen is formed can combine and will be completely dissociated, and after sample introduction, aptamers and target protein combine and form compound, Response (RU value) increases；Then terminate sample introduction, inject blank buffer solution, complex dissociation, the decline of RU value.

When screening active aptamers sequence using single-point method, after sample introduction, in the aptamers combinatorial libraries to Aptamers are screened in conjunction with the target protein for being fixed on chip surface, generate compound, sample introduction terminates, blank buffer solution is injected, Compound on chip dissociates, and obtains each sequence single point R U value change curve, and value (RU value) judges to be screened according to response Whether aptamers are active, and RU value is active greater than 0 aptamers.

For active aptamers sequence, then multi-cycle dynamic analysis (MCK analysis) is carried out, obtains affinity ginseng Number dissociation constant K_DAnd kinetic parameter, including association rate constant K_aWith dissociation rate constant K_d.Wherein K_D=K_d/K_a.Root According to K_DThe size of numerical value judges the affinity between aptamers to be screened and target protein, K_DNumerical value is smaller, and affinity is stronger, thus Filter out the strong aptamers sequence of affinity.

When carrying out single-point screening or carrying out MCK analysis, the aptamers to be screened in the aptamers combinatorial libraries exist Before sample introduction, prior to 90~98 DEG C (such as 95 DEG C) denaturation 5~10min (such as 5min), cooled to room temperature, then again into Sample.Injector temperature is 20~30 DEG C (such as 25 DEG C), and sample introduction binding time is 60~180 seconds (such as 120 seconds), and Dissociation time is 120~600 seconds (such as 300 seconds), flow velocity was 20~50 μ L/min (such as 30 μ L/min), and buffer used is Tris-T buffering Liquid.Regeneration condition is 5mM NaOH, and 30 μ L/min of flow velocity, the reproduction time is 15 seconds.

The wherein formula of the Tris-T buffer are as follows: 20mM Tris, 140mM NaCl, 5mM MgCl2,5mM KCl, Tween 20 is adjusted to pH7.4-7.6 with HCl.Wherein, 20 concentration range of Tween is 0.005%~0.1%, preferably 0.05%.

When carrying out single-point screening, each sequence sample introduction concentration in the stepping type aptamers combinatorial libraries can be 200 ~1000nM, such as 500nM.

In a specific embodiment, single-point screening can be achieved by step in detail below:

1) each sequence sample introduction concentration of each adaptor molecules group is 500nM.Each sequence is first in 95 DEG C, after 5min is denaturalized, Cooled to room temperature.The formula of buffer Tris-T buffer are as follows: 20mM Tris, 140mM NaCl, 5mM MgCl₂, 5mM KCl, 0.05%Tween 20 is adjusted to pH 7.4-7.6 with HCl；

2) SPR dynamic analysis experiment (n≤2) are carried out, the RU value according to every aptamers sequence filters out active Aptamers sequence, the aptamers sequence of value > 0 RU is active.When experiment, injector temperature selects 25 DEG C, sample introduction binding time 120s, Dissociation time 300s, 30 μ L/min of flow velocity；

3) regeneration condition can be the salt of high ionic strength, such as 1~3M NaCl；It may be acid condition, such as 10mM glycine, pH 1.5~2.5；It can also be alkaline condition, such as 1-15mM NaOH.It is preferred that regeneration condition is 5mM NaOH, reproduction time are set as 15 seconds.

When carrying out MCK analysis, can be selected according to RU value of the aptamers sequence to be analyzed in conjunction with the single-point of EPO- α Suitable concentration gradient range carries out MCK analysis.Such as the Stem-3bp of 500nM, RU value is lower, and only 3, it is selected dense Degree gradient then takes the circumstances into consideration to be selected as higher concentration range, such as 125nM~2000nM (being multiplication relation between each point)；And 500nM Stem-7bp, RU value are higher, are 80RU, the concentration gradient selected is 12.5nM~400nM.

In a specific embodiment, MCK analysis is achieved by step in detail below:

1) each sequence is first after 95 DEG C, 5min denaturation, cooled to room temperature.Combination buffer is Tris-T buffering Liquid.

2) MCK analysis is carried out, sample introduction binding time is set as 120s, Dissociation time 300s, 30 μ L/min of flow velocity；Regeneration condition For 5mM NaOH, 15 seconds.

3) nonlinear fitting is carried out to MCK result, obtains K_a, K_dAnd K_DValue.

When carrying out MCK analysis, need to also judge whether the nonlinear fitting result is normal from fitting result degree of conformity.Fitting As a result degree of conformity is by U value and Chi²Value determines.Xu≤15 U value Yi Ban illustrate that fitting degree of conformity is higher；U value is lower, and degree of conformity is got over Height, U value minimum 1；Chi²Yi Ban Xu≤0.1Rmax illustrates that fitting degree of conformity is higher.

Constructed stepping type aptamers combinatorial libraries are sieved using the screening technique of aptamers of the present invention Choosing, the results showed that

1) molecular group is shunk to the left, and only L37, L35, L33 are bioactive sequence, K_DValue be respectively 320 ± 30nM, 360 ± The feature gradually increased from low to high is presented in 110nM and 390 ± 30nM；

2) molecular group is shunk to the right, and only R37, R35, R33, R31 are bioactive sequence, K_DValue respectively 560 ± 170nM, The feature gradually decreased from high to low is presented in 1300 ± 150nM and 140 ± 40nM, 130 ± 30nM substantially；

3) molecular group is shunk at both ends, and In37, In35, In33, In31, In29, In27, In25 are active, but active difference It is larger.Its K_DValue is respectively 310 ± 120nM (In37), 390 ± 40nM (In35), 750 ± 30nM (In33), 390 ± 50nM (In31), 200 ± 1nM (In29), 52 ± 4nM (In27) and 2060 ± 125nM (In25), K_DIt is worth from In37 to In33 gradually It increases, is then gradually decreased again to In27, the K of In27_DValue reduction is the most obvious, then the K of In25_DValue sharply increases again, The affine activity of In23, In21 are basic to be lost；In 27 is the affine highest sequence of activity in all punctured sequences；

4) stem extends molecular group, and all 8 sequences are active, K_DValue be respectively 390 ± 50nM (Stem-2bp), 440±40nM(Stem-3bp)、80±10nM(Stem-5bp)、40±2nM(Stem-6bp)、30±5nM(Stem-7bp)、40 ±20nM(Stem-10bp),40±0.1nM(Stem-12bp),50±20nM(Stem-15bp).Wherein only Stem-2bp, The affine activity of Stem-3bp is lower, K_DValue is gradually decreased from 2bp up to 7bp, from 7bp to 15bp, K_DIt is worth basic one It causes.Highest affine activity is Stem-7bp.Meanwhile Stem-7bp, Stem-6bp, Stem-10bp, Stem-12bp, Stem- The affine activity of 15bp is superior to 39nt.

As can be seen that by commenting stepping type aptamers combinatorial libraries provided by the invention progress affinity and dynamics Valence can screen rapidly to obtain the aptamers sequence In 27 that affinity is stronger, sequence is shorter.

In addition, also may determine that aptamers sequence according to the affinity and dynamic characteristic of above-mentioned 4 groups of adaptor molecules groups In with the presence or absence of crucial recognition site.It shrinks in molecular group to the left, is only capable of forming higher structure G- tetramer structure (GQ) L37, L35, L33 there are affine recognition capabilities, and with the shortening of stem's structure, affine activity is also gradually reduced.

It shrinks in molecular group to the right, R37, R35, R33, the R31 for being also only capable of forming higher structure GQ have affine work Property, particularly, in the molecular group, sufficiently whether exposure has large effect to affine activity to 7A8A base.7A8A's is complete sudden and violent When revealing (R33) compared with masked (R35, R37), the activity of aptamers is basic to be restored.And the removal front and back 7A8A (R33, R31), it is living Property is essentially identical.

And both ends are shunk in molecular group, with the shortening of stem, affine activity is gradually decreased, and remains entire ring portion In27 affinity is restored completely, prompts to be likely to form new higher structure.

Present invention is alternatively directed to obtained most short aptamers In27, construct its rite-directed mutagenesis molecular group, with clearly specific Contribution of the base position to combination.The secondary sequence feature of In27 is only to remain the ring portion sequence of 39nt；Its primary structure sequence Column feature is, -- GG---------GGGG--GG--GGG；In conjunction with its circular dichroism spectra result, thus it is speculated that In27 has been likely to form with GQ For the tertiary structure of skeleton.

The base sequence of most short aptamers In27 is divided into GQ skeleton structure and 5 structural domains in addition to GQ skeleton (Domain).To construct each rite-directed mutagenesis molecular group, totally 7 groups, it is respectively designated as D1, D2, D3&4, D5, GQ and degeneracy sequence Column rite-directed mutagenesis molecular group, totally 31 sequences.Wherein:

D1 rite-directed mutagenesis molecular group includes: that 1A base is changed to C/T/G/X, 2A base and is changed to T/G and 1A2A base simultaneously It is changed to 7 sequences such as T base；

D2 rite-directed mutagenesis molecular group includes: that 6C base is changed to T/A/G/X, 8G base and is changed to T base, 6C8G while being changed to T Base, 11T base such as are changed to X (base deletion that X represents the site) and 10T11T base while being changed to X at 8 sequences；

D3&4 rite-directed mutagenesis molecular group includes: that 18T base is changed to A base and 23T base is changed to 2 sequences such as A base Column；

D5 rite-directed mutagenesis molecular group includes: that 27G base is changed to 3 sequences such as A/T/X；

GQ rite-directed mutagenesis molecular group includes: that 25G is changed to T/X (base deletion that X represents the site), i.e. 25T, 25X；6 Under the premise of the C of position is changed to T, the G base in 14G, 15G, 16G, 17G is changed to T base, as 14T, 15T, 16T, 17T respectively, And two G bases in 14G15G, 15G16G, 16G17G, 14G17G are changed to T base, i.e. 14T15T, 15T16T, 10 sequences such as 16T17T, 14T17T；

Degenerate sequence molecular group includes 1A6C base while being changed to T base, i.e. 1T6T, 1A6C27G base is changed to respectively 1T16T27A base and 1A27G base are changed to 3 sequences such as 1T27A base.

Using single-point screening method, its affine active screening and evaluation are carried out to the rite-directed mutagenesis molecular group.

The result shows that GQ skeleton is extremely important to identifying, 25G takes part in the formation of GG skeleton in GQ, 14-17 G alkali Base may be related to GQ skeleton；D2 structural domain is the main domains that most short aptamers In27 and EPO- α interact； Sequence conservation on D2, D3, D4 ring is higher, and in addition to 6C, other bases cannot be changed arbitrarily.The results show that the height of In27 Spend conservative.

To which its degenerate sequence can be obtained are as follows:

¹NAGGT⁶YTGT¹⁰TTTTGGGGTT²⁰GGTTTGGG

Wherein, N represents A, G, T or C, and Y represents C or T.

Advantageous effects of the invention include following one or more aspects:

1) method of building aptamers combinatorial libraries provided by the invention, is constructed using efficient structural tailoring mode and is included The combinatorial libraries of less sequence, constructed aptamers combinatorial libraries cover the most of possibility that can generate special affinity interaction Range to obtain most short aptamers, and inquires into possible binding mechanism and lays a good foundation.

2) aptamers screening technique provided by the invention is screened using aptamers combinatorial libraries provided by the invention and is adapted to Body can filter out the most short aptamers bioactive sequence of more excellent, the irredundant sequence of affinity, and can define in specific aptamers The effect in critical base site.

3) aptamers screening technique provided by the invention is applied, screens and has obtained affinity more preferably sequence: Stem-7bp, Stem-6bp, Stem-10bp, Stem-12bp, Stem-15bp, In27, wherein In27 is the most short aptamers of irredundant sequence Bioactive sequence.These sequences can be used for detecting epo protein.

4) most short aptamers sequence In27 obtained for screening, it is prominent using fixed point base in conjunction with the feature of the sequence Become, illustrates contribution of the critical base to combination.

5) degenerate sequence of most short aptamers In27 is obtained.In27 and its degenerate sequence are provided with good between EPO- α Affine activity is suitable as affine sensing function element.

Detailed description of the invention

The Sequence composition figure of Fig. 1 stepping type aptamers combinatorial libraries；

Each rite-directed mutagenesis molecular group Sequence composition figure of Fig. 2；

When the amino acid residue end of Fig. 3 A.EPO- α is amino group, the reacting flow chart with biotin covalent coupling；

When the amino acid residue end of Fig. 3 B.EPO- α is carboxylic group, the reacting flow chart with biotin covalent coupling；

When the glycosyl end of Fig. 3 C.EPO- α is sialic acids groups, the reacting flow chart with biotin covalent coupling；

Fig. 4 fixes the combining response of the aptamers sequence (500nM, 39nt) after Bio-EPO- α (1 μM) on SA chip, Wherein:

It (A) is to fix Bio-NH on SA chip₂RU value change curve when-EPO- α, RU_stability=2342, it is fixed Condition: sample introduction flow velocity is 10 μ L/min, and sample injection time is 60 seconds；

(B) Bio-NH is evaluated as analyte for 500nM 39nt₂RU value change curve when the fixed effect of-EPO；

It (C) is the RU value change curve when fixing Bio-Sialyl-EPO- α on SA chip, RU_stability=2342, Gu Fixed condition: sample introduction flow velocity is 10 μ L/min, and sample injection time is 60 seconds；

(D) Sialyl-NH is evaluated as analyte for 500nM 39nt₂RU value when the fixed effect of-EPO changes bent Line；

Bio-NH is fixed on Fig. 5 A.SA chip₂When-EPO- α, spectrogram is dissociated in the combination of 39nt；

When fixing Sialyl-Bio-EPO- α on Fig. 5 B.SA chip, spectrogram is dissociated in the combination of 39nt；

Fig. 6 shrinks the single point R U value change curve of each sequence of adaptor molecules group to the left；

Fig. 7 shrinks to the right the single point R U value change curve of each sequence of adaptor molecules group；

The single point R U value change curve of the both ends Fig. 8 contraction each sequence of adaptor molecules group；

The single point R U value change curve of each sequence of Fig. 9 both ends extension adaptor molecules group；

Figure 10 is In27 in K containing various concentration⁺Tris-HCl buffer in circular dichroism spectrogram；

In Figure 11 A.GQ rite-directed mutagenesis adaptor molecules group, the rite-directed mutagenesis single-point the selection result of 14-17 G bases, Reference sequence is In27, and every sequence concentration is 500nM；

The partial enlarged view of Blocked portion in Figure 11 B. Figure 11 A, it can be seen that the rite-directed mutagenesis single-point screening of 25 G bases As a result；

The single-point the selection result of each sequence of Figure 12 .D1 rite-directed mutagenesis adaptor molecules group, reference sequence In27, every sequence Column concentration is 500nM；

The single-point the selection result of each sequence of Figure 13 .D2 rite-directed mutagenesis adaptor molecules group, reference sequence In27, every sequence Column concentration is 500nM；

The single-point the selection result of each sequence of Figure 14 .D3&4 rite-directed mutagenesis adaptor molecules group, reference sequence In27, every Sequence concentration is 500nM；

The single-point the selection result of each sequence of Figure 15 .D5 rite-directed mutagenesis adaptor molecules group, reference sequence In27, every sequence Column concentration is 500nM；

The single-point the selection result of each sequence of Figure 16 degenerate sequence adaptor molecules group, reference sequence In27, every sequence Concentration is 500nM；

Figure 17 A. is when fixing 3 '-Biotin-39nt on SA chip, the MCK spectrogram of EPO- α, and every sequence concentration is 500nM；

Figure 17 B. is when fixing 3 '-Biotin-12T-In27 on SA chip, the MCK spectrogram of EPO- α, every sequence concentration For 500nM.

Specific embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific Condition person, the condition suggested according to normal conditions with manufacturer carry out, and reagents or instruments used without specified manufacturer is The product of commercially available acquisition can be passed through.

Affinity between aptamers and target molecule is characterized and evaluated in the embodiment of the present invention using SPR method.Specifically, made Instrument is BIAcore T100 interaction of biomacromolecules instrument, is purchased from U.S. GE company.

When experiment, sample flows through chip surface with constant flow velocity and concentration, analysans in sample (such as the present invention Aptamers in constructed aptamers stepping type combinatorial libraries) and it is fixed on molecule (such as the NH of chip surface₂-Bio- EPO- α or Sialyl-Bio-EPO- α) occur in conjunction with compound is formed, the quality of chip surface substance changes, instrument note The variation of corresponding response (Response Unit, hereinafter referred to as RU value) under record.After terminating sample introduction, make plain buffer stream Chip surface is crossed, compound dissociates, and the quality of chip surface substance changes again, the corresponding RU value of instrument record Variation.

The technology can track and record analysans in sample in conjunction with the molecule of chip surface, dissociation whole process Variation records the variation of RU value, generates combination-dissociation curve.Pass through multi-cycle dynamics (Multiple Cycle Kinetics, MCK) analysis, moreover it is possible to dynamics data is provided (for example, association rate constant K_a, dissociation rate constant K_d) and parent With force data (for example, dissociation constant K_D, K_D=K_d/K_a)。

After being coupled in conjunction with the molecule of chip surface, the aptamers sequence of value > 0 RU has aptamers sequence to be analyzed Activity.For active aptamers sequence, MCK analysis, the parent between available each aptamers sequence and EPO- α are carried out With power and kinetic parameter.Affinity is with dissociation constant K_DCharacterization, K_DLower, then affinity is higher.In kinetic parameter, in conjunction with Rate constant K_aIndicate that the speed that compound is formed between analyte and ligand molecular, numerical value are equivalent to the analyte and ligand of 1M The quantity of the compound formed when mixing, unit M^-1s^-1；Dissociation rate constant K_dIndicate the speed of complex dissociation, numerical value It is equivalent to the ratio of compound dissociation per second, unit s^-1).According to active different, the K of each aptamers and EPO- α_DValue generally exists NM~μM range.

Buffer used in the embodiment of the present invention is as follows:

1) 5 × PBS buffer solution, formula are as follows: 0.75M NaCl, 0.1M Na₃PO₄, pH value 7.2；

2) 1 × PBS buffer solution, formula are as follows: 0.15M NaCl, 0.02M Na₃PO₄, pH value 7.2；

3) 5 × Tris-HCl buffer, formula are 100mM Tris, 700mM NaCl, 25mM MgCl₂, 25mM KCl, pH value 7.4-7.6；

4) Tris-T buffer, formula are 20mM Tris, 140mM NaCl, 5mM MgCl₂, 5mM KCl, 0.05wt%Tween 20 is adjusted to pH 7.4-7.6 with HCl.

The building of 1 aptamers stepping type combinatorial libraries of embodiment

As shown in Figure 1, selecting the single stranded DNA aptamers of EPO- α shown in SEQ ID NO:1 as initiation sequence, respectively Building shrinks molecular group to the left, shrinks molecular group, both ends contraction molecular group and stem's extension molecular group to the right, by this four molecules Group collectively forms aptamers stepping type combinatorial libraries.

1) building of molecular group is shunk to the left

On the basis of initiation sequence 39nt, punctures two adjacent bases step by step from its 5 ' end, obtain as in table 1 Shown in Left contraction 37,35,33 ..., until Left contraction 21 (respectively abbreviation L37, L35, L33 ..., L21) etc. 9 sequences.

2) building of molecular group is shunk to the right

It is then exactly the opposite that molecular group is shunk to the right, on the basis of initiation sequence 39nt, is punctured step by step from its 3 ' end Two adjacent bases, obtain Right contraction as shown in table 1 37,35,33 ..., until Right Contraction 21 (respectively abbreviation R37, R35, R33 ..., R21) etc. 9 sequences.

3) building of molecular group is shunk at both ends

It is to cut out step by step respectively on the basis of initiation sequence 39nt from its 5 ' and 3 ' end two sides that molecular group is shunk at both ends Fall a base, obtain Inner contraction as shown in table 1 37,35,33 ..., until Inner Contraction21 (respectively abbreviation In37, In35, In33 ..., In21) etc. 9 sequences.

4) stem extends the building of molecular group

Stem extend molecular group be then keep aptamers ring portion sequence it is constant in the case where, stem's sequence be changed to include Second sequence complementary base of the First ray ... of G and A comprising C and T, wherein First ray be respectively as follows: GA, AGA, AGAGA, GAGAGA, AGAGAGA, GAGAGAGAGA, GAGAGAGAGAGA, AGAGAGAGAGAGAGA, the second sequence be respectively as follows: TC, TCT, TCTCT,TCTCTC,TCTCTCT,TCTCTCTCTC,TCTCTCTCTCTC,TCTCTCTCTCTCTCT.It obtains as shown in table 1 Stem-2bp, Stem-3bp, Stem-5bp, Stem-6bp, Stem-7bp, Stem-10bp, Stem-12bp, Stem-15bp 8 sequences such as (being briefly referred to as S2bp, S3bp, S5bp, S6bp, S7bp, S10bp, S12bp, S15bp).

Aptamers stepping type combinatorial libraries constructed by the present embodiment have only used 35 sequences, that is, spy can be generated by covering Most of possible ranges of different affinity interaction.Aptamers are screened using above-mentioned stepping type combinatorial libraries, can determine that specificity is suitable The site of critical base in ligand, to obtain that affine activity is more excellent, the stronger optimization aptamers sequence of specificity.

Above-mentioned aptamers stepping type combinatorial libraries are synthesized by Shanghai Sheng Gong bio-engineering corporation.

The sequence table of 1 stepping type combinatorial libraries of table

When the amino acid residue end of embodiment 2EPO- α is amino group, the covalent coupling with biotin

When the amino acid residue end of EPO- α is amino group, reaction process such as Fig. 3 A institute with biotin covalent coupling Show.

Weigh coupling reagent (Sulfo-NHS-LC-Biotin, be purchased from U.S. Thermo company) 0.35mg, 252 μ L 5 × PBS buffer solution, 1008 μ L sterilizing ultrapure water, is configured to the 0.5mM Sulfo-NHS-LC-Biotin solution of 1260 μ L, the solution Matching while using, and it is protected from light operation.Take EPO- α albumen stoste (3.25g/L, Shenzhen match Bao Er's biology Pharma Inc. of 15.5 μ L Give), 20 5 × PBS of μ L and 64.5 μ L sterilizing ultrapure water is added, is configured to the 0.5g/L EPO- α solution of 100 μ L.To this 100 In the EPO- α solution of μ L 0.5mg/mL, the 0.5mM Sulfo-NHS-LC-Biotin solution of 6.25 μ L is added, after mixing, in Shaken at room temperature incubates 30min.It after the completion of incubation reaction, is purified, reaction solution is added to desalination pillar (such as Zeba Spin Desalination pillar is purchased from Thermo company) in, remove remaining non-reacted molecules.Repeat purifying 2 times.

By solution centrifugal concentrating after purification to 50 μ L, and ultraviolet-visible spectrophotometry is utilized, with HABA/Avidin method Characterization, the label number for obtaining biotin are 1 to get having arrived the biotinylated NH of unimolecule₂-Bio-EPO-α.Use UV, visible light Spectrophotometry is to the NH₂The concentration of-Bio-EPO- α is measured, concentration calculation formula are as follows: and protein concentration (mg/mL)= 1.55A₂₈₀-0.76A₂₆₀), the concentration being calculated is 1.04g/L, the rate of recovery 103%.

Covalent coupling when the amino acid residue terminal carboxyl groups of embodiment 3EPO- α, with biotin

When the amino acid residue end of EPO- α is carboxylic group, reaction process such as Fig. 3 B institute with biotin covalent coupling Show.

Weigh coupling reagent (Biotin Hydrazide is purchased from U.S. Sigma Aldrich) 0.34mg, 229 μ L Sterilize ultrapure water, is configured to 4mM Biotin Hydrazide solution, the solution matching while using, and be protected from light operation.Take 4mM 0.5M 2- (N- morpholine)-ethanesulfonic acid solution (MES solution, pH=of 100 μ L is added in 50 μ L of Biotin Hydrazide solution 5.0) sterilize ultrapure water with 350 μ L, is configured to 0.4mM Biotin Hydrazide solution, and buffer system is 0.1M MES (pH 5.2).Carbodiimide (EDC) 5.57mg is weighed, 1166 μ L sterilizing ultrapure water is added and is configured to 25mM EDC solution.Take 40 μ L's The 0.5M MES solution (pH=5.0) of 200 μ L is added in 25mM EDC solution, and it is molten that 760 μ L sterilizing ultrapure water is configured to 1mM EDC Liquid, buffer system are 0.1M MES (pH 5.2).The EPO- α albumen stoste (3.25g/L) of 15.5 μ L is taken, is added 20 μ L's 0.5M MES buffer and 64.5 μ L sterilizing ultrapure water are formulated as the 0.5g/L EPO- α solution of 100 μ L.To the 100 μ L 0.5g/ In the EPO- α solution of L, the 1mM EDC solution of 0.4mM Biotin Hydrazide solution and 15 μ L that 7.5 μ L are added is mixed Afterwards, 2h is incubated in shaken at room temperature.It after the completion of incubation reaction, is purified, reaction solution is added to desalination pillar (such as Zeba Spin desalination pillar is purchased from Thermo company) in, remaining non-reacted molecules are removed, and reaction buffer system is changed into PBS and is delayed Fliud flushing.Purifying 2 times is repeated, solution after purification is obtained.

By solution centrifugal concentrating after purification to 50 μ L, and ultraviolet-visible spectrophotometry is utilized, with HABA/Avidin method Characterization, the label number for obtaining biotin are 1 to get having arrived the biotinylated COOH-Bio-EPO- α of unimolecule.With it is ultraviolet can See that spectrophotometry is measured the concentration of the COOH-Bio-EPO- α, concentration calculation formula is calculated with embodiment 1 Concentration is 1.07g/L, the rate of recovery 109%.

When the glycosyl end of embodiment 4EPO- α is sialic acids groups, the covalent coupling with biotin

It is as shown in Figure 3 C with the reaction process of biotin covalent coupling when the glycosyl end of EPO- α is sialic acids groups.

It is protected from light and weighs 8.96mg sodium metaperiodate (NaIO₄), 873.8 μ L sterilizing ultrapure water is added, obtains final concentration of 50mM NaIO₄Stock solution takes the 50mM NaIO of 4 μ L₄0.5M sodium acetate (NaAc) solution (pH=5.5) of 40 μ L is added in stock solution, 156 μ L sterilizing ultrapure water is configured to 1mM NaIO₄Solution, buffer system are 0.1M NaAc (pH5.5).Take the EPO- α of 15.5 μ L Albumen stoste (3.25g/L), is added 20 μ L 0.5M NaAc solution and 64.5 μ L sterilizing ultrapure water is configured to the 0.5g/L of 100 μ L EPO- α solution.Into the 0.5g/L EPO- α solution of the 100 μ L, the 1mM NaIO of 2.35 μ L is added₄Solution mixes, and keeps away in 4 DEG C Light generation incubates 30min.It after the completion of incubation reaction, is purified, reaction solution is added to desalination pillar, and (such as Zeba Spin is de- Salt pillar is purchased from Thermo company) in, remaining non-reacted molecules are removed, and reaction buffer system is changed into PBS buffer solution. Purifying 2 times is repeated, EPO- alpha reaction liquid after purification is obtained.

It is protected from light and weighs 0.31mg coupling reagent Biocytin Hydrazide, the PBS buffer solution of 333.8 μ L is added, obtains The Biocytin Hydrazide solution of final concentration of 2.5mM.Into EPO- alpha reaction liquid after purification, it is added 4.7 μ L's 2.5mM Biocytin Hydrazide solution mixes, is protected from light incubated under agitation at room temperature 2 hours.After the completion of incubation reaction, into Reaction solution is added in desalination pillar (such as Zeba Spin desalination pillar is purchased from Thermo company), removes remaining by row purifying Non-reacted molecules.Purifying 2 times is repeated, solution after purification is obtained.

By solution centrifugal concentrating after purification to 50 μ L.And ultraviolet-visible spectrophotometry is utilized, with HABA/Avidin method Characterization, the label number for obtaining biotin are 1 to get having arrived the biotinylated Sialyl-Bio-EPO- α of unimolecule.With ultraviolet Visible spectrophotometry is measured the concentration of the Sialyl-Bio-EPO- α, and concentration calculation formula is calculated with embodiment 1 The concentration arrived is 1.38mg/mL, the rate of recovery 137%.

NH on embodiment 5SA chip₂The fixation of-Bio-EPO- α

Take the NH of 2.7 μ L₂- Bio-EPO- α stock solution (stock solution is configured with ultrapure water, and concentration is 36.5 μM), is added 5 × Tris-HCl buffer of 20 μ L and 77.3 μ L purified waters are to get to 1 μM of NH₂- Bio-EPO- α solution.

One piece of new SA chip is taken, first with 50mM NaOH, 1M NaCl solution carries out channel initialization.Sample introduction 3 times, Flow velocity is 20 μ L/min, and the time of each sample introduction is 60 seconds.Then place is normalized to SA chip with 70% glycerine water solution Reason.

Sample introduction under conditions of flow velocity is 10 μ L/min, sample injection time is 60 seconds, by 1 μM of NH₂- Bio-EPO- α (preparation Method is shown in embodiment 2) it is fixed on the channel 2 of SA chip.During sample introduction, Bio-NH₂- EPO- α and SA chip form biology The strong Non-covalent binding of element-Streptavidin, RU value rise.At the end of sample introduction, RU value rises to maximum value.Then slow with blank again Fliud flushing rinses SA chip surface, except the Bio-NH of attachment removal₂- EPO- α, RU value can be declined slightly.RU value change curve is referring to fig. 4 In (A), the maximum RU value after rising subtract decline after RU value be to get to response stability (stability) RU value 2342, that is, RU_stability=2342.

It is reference channel with channel 1, using the 39nt of 500nM, to Bio-NH fixed on channel 2₂- EPO- α albumen Activity is evaluated.The flow velocity of sample introduction is 30 μ L/min, and sample injection time is 120 seconds.After sample introduction, 39nt and Bio-NH₂-EPO-α Albumen forms Non-covalent binding, and RU value rises.At the end of sample introduction, RU value rises to maximum value.Then it is rushed again with plain buffer Wash SA chip surface, the dissociation of Non-covalent binding object, the decline of RU value.RU value change curve (combine-dissociation curve) referring to fig. 4 in (B), 39nt and Bio-NH₂During-EPO- α protein binding, it is corresponding stability that RU maximum value, which subtracts RU minimum value, The response stability Δ RU in the channel Δ RU, 2-1 is 30.Illustrate under the fixed form, maintains affine work between EPO- α and 39nt Property.

Regeneration condition is 5mM NaOH, and 30 μ L/min of flow velocity, the reproduction time is 15 seconds.

The fixation of Sialyl-Bio-EPO- α on embodiment 6SA chip

The Sialyl-Bio-EPO- α stock solution (stock solution is configured with ultrapure water, and concentration is 54.3 μM) for taking 1.8 μ L, adds Enter 5 × Tris-HCl buffer and the 78.2 μ L purified waters of 20 μ L to get to 1 μM of Sialyl-Bio-EPO solution.

One piece of new SA chip is taken, channel initialization is carried out with 50mM NaOH, 1M NaCl solution first.Sample introduction 3 times, Flow velocity is 20 μ L/min, and the time of each sample introduction is 60 seconds.Then chip is normalized with 70% glycerine water solution.

Sample introduction under conditions of flow velocity is 10 μ L/min, sample injection time is 60 seconds, by 1 μM of Sialyl-Bio-EPO- α (preparation method is shown in embodiment 4) is fixed in SA chip channel 4.RU value change curve referring to fig. 4 in (C), response stability (stability) RU value rises 1159, that is, RU_stability=1159.

It is reference channel with channel 3, using the 39nt of 500nM, to Sialyl-Bio-EPO- α albumen fixed on channel 4 Activity evaluated.The flow velocity of sample introduction is 30 μ L/min, and sample injection time is 120 seconds.RU value change curve referring to fig. 4 in (D), the response stability Δ RU in the channel 4-3 is 50.Illustrate under the fixed form, still maintains between EPO- α and 39nt affine Activity.

Regeneration condition is 5mM NaOH, and 30 μ L/min of flow velocity, the reproduction time is 15 seconds.

The MCK of 7 39nt of embodiment is analyzed

100 μM of 39nt stock solutions (stock solution is configured with ultrapure water) of 2 μ L are taken, 5 × Tris-HCl that 4 μ L are added is slow Fliud flushing, the 1wt%Tween 20 of 2 μ L and 12 μ L sterilizing ultrapure water, obtain 10 μM of 39nt solution.Take 20 μ of the 39nt solution The Tris-T buffer of 380 μ L is added to get the 39nt solution of 500nM is arrived in L.Using the side of Tris-T buffer doubling dilution The 39nt solution that concentration is respectively 25,50,100,200,400nM is prepared in method, is denaturalized 5 minutes at 95 DEG C, then naturally cold But to room temperature.

On SA chip, NH is used₂- Bio-EPO- α (channel 2) and Sialyl-Bio-EPO- α (channel 4) carries out EPO- Affinity and dynamic analysis between α and 39nt.Binding time is 120 seconds, and Dissociation time is 300 seconds, and flow velocity is 30 μ L/min. Regeneration condition is 5mM NaOH, and sample injection time is 15 seconds, and flow velocity is 30 μ L/min.Comprising using blank solvent as zero point including The Loading sequence of six concentration of 39nt is 0,25nM, 50nM, 100nM, 200nM, 400nM, obtains combining dissociation spectrogram, such as Fig. 5 A With shown in Fig. 5 B, wherein Fig. 5 A is (the fixed NH of channel 2₂- Bio-EPO- α) obtain combination dissociation spectrogram, Fig. 5 B be channel 4 The combination dissociation spectrogram that (fixed Sialyl-Bio-EPO- α) is obtained.

Nonlinear fitting is carried out in conjunction with dissociation spectrogram to shown in Fig. 5 A and Fig. 5 B using Biacore Data Analysis Software. The result shows that obtaining the dissociation constant K of 39nt and EPO- α interaction using channel 2-1 data_D=55nM, association rate are normal Number K_a=1.6 × 10⁴M^-1s^-1, dissociation rate constant K_d=8.9 × 10^-4s^-1(U-value=1, Chi²=0.13).Utilize channel 4-3 data obtain the dissociation constant K of 39nt and EPO- α interaction_D=52nM, association rate constant K_a=1.8 × 10⁴M^-1s^-1, dissociation rate constant K_d=9.3 × 10^-4s^-1(U-value=1, Chi²=0.07).The above results show with NH₂-Bio- When EPO- α and Sialyl-Bio-EPO- α are fixed form, the two degree of conformity is preferable, also illustrates that SPR method is successfully built It is vertical.

Compared with Sialyl-Bio-EPO- α, NH₂- Bio-EPO- α is higher to the tolerance that 5mM NaOH is regeneration condition.

Embodiment 8 shrinks MCK points of single site ac tive evaluation and bioactive sequence of the adaptor molecules group on SA chip to the left Analysis

Aptamers sequence to be evaluated are as follows: all sequences in contraction adaptor molecules group to the left, including L21, L23, L25, L27, L29, L31, L33, L35, L37 and reference sequence 39nt, totally 10 sequences.Above-mentioned 10 sequences of 2 μ L are measured respectively The stock solution (stock solution is configured with ultrapure water, and concentration is 100 μM) of column, is added 5 × Tris-HCl buffer of 4 μ L, 2 μ L's 20,12 μ L of 1wt%Tween sterilizing ultrapure water, obtains stock solution among 10 μM of each sequence.Take storage among each sequence of 20 μ L Standby liquid (10 μM) are added the Tris-T buffer of 380 μ L, obtain each sequence operative solution of 500nM, be denaturalized at 95 DEG C 5min, cooled to room temperature.

Using securing NH₂The SA chip of-Bio-EPO- α, with shrinking in adaptor molecules group to the left for the 500nM Each sequence and reference sequence 39nt carry out SPR dynamic analysis using single-point screening method as analyte.Specific experiment condition With embodiment 7.After sample introduction, analysans generates compound in conjunction with the EPO- α on SA chip, and sample introduction terminates, and injection blank is slow Solution is rushed, the compound on chip dissociates, and each sequence single point R U value change curve is as shown in Figure 6.

The result shows that L37, L35, L33 for being only capable of forming the higher structure G- tetramer (hereinafter referred to as GQ) structure are deposited In affine recognition capability, and with the shortening of stem's structure, response is also gradually reduced.

For this three bioactive sequences of L37, L35, L33, choosing concentration respectively is 31.25-1000nM (L37)；62.5- 2000 nM(L35)；62.5-2000nM (L33) carries out MCK analysis.Specific SPR condition is the same as embodiment 7.It obtains affinity and moves Mechanics parameter, as shown in table 2.The result shows that K_DValue is respectively 320 ± 30nM, 360 ± 110nM and 390 ± 30nM, is presented The feature gradually increased from low to high.

Table 2 shrinks the affinity and kinetic parameter of adaptor molecules group to the left

Example 9 shrinks to the right the MCK analysis of single site ac tive evaluation and bioactive sequence of the adaptor molecules group on SA chip

Aptamers sequence to be evaluated are as follows: all sequences in contraction adaptor molecules group to the right, including R21, R23, R25, R27, R29, R31, R33, R35, R37 and reference sequence 39nt, totally 10 sequences.Above-mentioned 10 sequences of 2 μ L are measured respectively The stock solution (stock solution is configured with ultrapure water, and concentration is 100 μM) of column, is added 5 × Tris-HCl buffer of 4 μ L, 2 μ L's 20,12 μ L of 1wt%Tween sterilizing ultrapure water, obtains stock solution among 10 μM of each sequence.Take storage among each sequence of 20 μ L Standby liquid (10 μM) are added the Tris-T buffer of 380 μ L, obtain each sequence operative solution of 500nM, be denaturalized at 95 DEG C 5min, cooled to room temperature.

Using securing NH₂The SA chip of-Bio-EPO- α, with shrinking in adaptor molecules group to the right for the 500nM Each sequence and reference sequence 39nt are analyte, carry out SPR dynamic analysis using single-point screening method.Specific experiment condition is same Embodiment 7.After sample introduction, analysans generates compound in conjunction with the EPO- α on SA chip, and sample introduction terminates, injection blank buffering Solution, the compound on chip dissociate, and each sequence single point R U value change curve is as shown in Figure 7.

The result shows that R37, R35, R33, R31 for being only capable of forming higher structure GQ have affine activity, particularly, In the molecular group, sufficiently whether exposure has large effect to response to the base in the site 7A8A.The base in the site 7A8A is complete When full exposure (R33) is compared with masked (R35, R37), the activity of aptamers is basic to be restored.And before the base removal in the site 7A8A (R33) and after removal (R31), response is essentially identical.

For this three bioactive sequences of R37, R35, R33, R31, choosing concentration respectively is 31.25-1000nM (R37)； 125-4000nM(R35)；31.25-1000nM(R33)；31.25-1000nM (R31) carries out MCK analysis.Specific SPR condition is same Embodiment 7.Affinity and kinetic parameter are obtained, as shown in table 3.The result shows that K_DValue is respectively 560 ± 170nM, 1300 The feature gradually decreased from high to low is presented in ± 150nM, 140 ± 40nM and 130 ± 30nM.

Table 3 shrinks to the right the affinity and kinetic parameter of adaptor molecules group

MCK points of single site ac tive evaluation and bioactive sequence of the 10 both ends extension adaptor molecules group of embodiment on SA chip Analysis

Aptamers sequence to be evaluated are as follows: all sequences in both ends extension adaptor molecules group, including Stem-2bp, Stem-3bp, Stem-5bp, Stem-6bp, Stem-7bp, Stem-10bp, Stem-12bp, Stem-15bp and reference sequence 39nt is arranged, totally 10 sequences.The stock solution (concentration of stock solution is 100 μM) for measuring above-mentioned 10 sequences of 2 μ L respectively, is added 4 μ 5 × Tris-HCl buffer of L, 20,12 μ L of the 1wt%Tween sterilizing ultrapure water of 2 μ L, obtains 10 μM of each sequence deposit Liquid.Each sequence stock solution (10 μM) of 20 μ L is taken, the Tris-T buffer of 380 μ L is added, each sequence operative for obtaining 500nM is molten Liquid is denaturalized 5min, cooled to room temperature at 95 DEG C.

Using securing NH₂The SA chip of-Bio-EPO- α, in the both ends extension adaptor molecules group of the 500nM Each sequence and reference sequence 39nt are analyte, carry out SPR dynamic analysis using single-point screening method.Specific experiment condition is same Embodiment 7.After sample introduction, analysans generates compound in conjunction with the EPO- α on SA chip, and sample introduction terminates, injection blank buffering Solution, the compound on chip dissociate, and each sequence single point R U value change curve is as shown in Figure 8.

The result shows that, with the extension of stem, response gradually rises from Stem-2bp to Stem-7bp.And from Stem- 7bp is until Stem-15bp, response are then gradually reduced.In addition to Stem-5bp, the response of other sequences is above positive sequence Arrange 39nt.And affine active maximum enhancing comes across Stem-7bp.

For all sequences in both ends extension adaptor molecules group, choose respectively concentration be 31.25-1000nM (2bp), 125-4000nM(3bp)、31.25-500nM(5bp)、31.25-500nM(6bp)、31.25-500nM(7bp)、31.25- 500nM (10bp), 31.25-500nM (12bp), 31.25-500nM (15bp) carry out MCK analysis.Specific SPR condition is the same as implementation Example 7.Affinity and kinetic parameter are obtained, as shown in table 4.The result shows that K_DValue from Stem-2bp until Stem-7bp by Gradually reduce, so from Stem-7bp to Stem-15bp, K_DValue flattens out substantially.Highest affine activity is Stem-7bp.Meanwhile The affine activity of Stem-7bp, Stem-6bp are superior to 39nt.

The affinity and kinetic parameter of 4 both ends extension adaptor molecules group of table

Shrink MCK points of single site ac tive evaluation and bioactive sequence of the adaptor molecules group on SA chip in 11 both ends of embodiment Analysis

Aptamers sequence to be evaluated are as follows: all sequences in both ends contraction adaptor molecules group, including In21, In23, In25, In27, In29, In31, In33, In35, In37 and reference sequence 39nt, totally 10 sequences.It is measured on 2 μ L respectively The stock solution (stock solution is configured with ultrapure water, and concentration is 100 μM) for stating 10 sequences, is added 5 × Tris-HCl buffering of 4 μ L Liquid, 20,12 μ L of the 1wt%Tween sterilizing ultrapure water of 2 μ L, obtains stock solution among 10 μM of each sequence.Take each sequence of 20 μ L Intermediate stock solution (10 μM) is arranged, the Tris-T buffer of 380 μ L is added, each sequence operative solution of 500nM is obtained, at 95 DEG C It is denaturalized 5min, cooled to room temperature.

Using securing NH₂The SA chip of-Bio-EPO- α is shunk in adaptor molecules group with the both ends of the 500nM Each sequence and reference sequence 39nt are analyte, carry out SPR dynamic analysis using single-point screening method.Specific experiment condition is same Embodiment 7.After sample introduction, analysans generates compound in conjunction with the EPO- α on SA chip, and sample introduction terminates, injection blank buffering Solution, the compound on chip dissociate, and each sequence single point R U value change curve is as shown in Figure 9.

The experimental results showed that response gradually decreases, and the In27 for remaining entire ring portion is affine with the shortening of stem Power is restored completely, prompts to be likely to form new higher structure.

For this seven bioactive sequences of In37, In35, In33, In31, In29, In27, In25, choosing concentration respectively is 31.25-1000nM(In37、In31)、125-4000nM(In35、In33、In25)、62.5-2000nM(In29)、12.5- 400nM (In27) carries out MCK analysis.Specific SPR condition is the same as embodiment 7.Affinity and kinetic parameter are obtained, such as 5 institute of table Show.The result shows that K_DValue gradually rises from In37 to In33, then gradually decreases again, the K of In27_DValue reduction is the most obvious, and The K of In25 afterwards_DValue sharply increases again, and In 27 is the affine highest sequence of activity in all punctured sequences.

The affinity and kinetic parameter of 5 both ends of table contraction adaptor molecules group

The higher structure that embodiment 12 is likely to form using circular dichroism spectra measurement In27

The In27 freeze-dried powder of 0.6OD (0.6OD is equivalent to 0.6*33 μ g=19.8 μ g) is taken to be dissolved in containing different dense respectively Spend K⁺Three kinds of Tris-HCl (20mM Tris, 140mM NaCl, 5mM MgCl₂, pH 7.4-7.6 is adjusted to HCl) and buffer In, wherein KCl is respectively 0,5,100mM.Set temperature is 25 DEG C, carries out circular dichroism spectra measurement, obtains circle as shown in Figure 10 Two chromatograms.Instrument is MOS-450 Multi-functional circular dichroscope spectrometer, French BioLogic Science Instruments Company, colorimetric pool light path 1cm.

It can be seen that from circular dichroism spectrogram shown in Fig. 10 in various concentration K⁺Tris-HCl buffer in, In27 is equal Negative peak is formd at 260nm, posivtive spike is formd at 290nm, meets the higher order conformation feature of the antiparallel G- tetramer (GQ). Illustrate that In27 forms antiparallel G tetramer structure in Tris-HCl buffer.K⁺Promote the G- tetramer advanced with induction The effect that structure is formed, but as can be seen that K in the circular dichroism spectrogram⁺Concentration be 0 and 5mM when, be formed by spectrogram and almost weigh It closes, illustrates that In27 itself can form the G- tetramer, and the higher structure is relatively stable, K⁺Advanced knot of the presence or absence to In27 Structure influences not significant.

The rite-directed mutagenesis adaptor molecules group of the building of embodiment 13 In27

For most short aptamers In27, its rite-directed mutagenesis molecular group is constructed, with clear In27 in conjunction with EPO- alpha specific When, the contribution of specific base position.

The secondary sequence feature of In27 are as follows: the ring portion sequence of 39nt is only remained, primary structure sequence signature is, -- GG---------GGGG--GG--GGG；In conjunction with its circular dichroism spectra result, thus it is speculated that In27 has been likely to form three using GQ as skeleton Level structure.

In addition to the GG skeleton on In27, other sequences are interval by-GG (G)-, are divided into D1~5 structural domain (Domain), rite-directed mutagenesis is carried out.As shown in Fig. 2, construct each rite-directed mutagenesis molecular group, totally 6 groups, be respectively designated as GQ, D1, D2, D3&4, D5 and degenerate sequence rite-directed mutagenesis molecular group, totally 31 sequences.Being mutated the big principle followed is: the examination based on experience Wrong method enables the optimization aptamers to have wider applicability its object is to find degenerate sequence.In view of G-C is matched, C base is not introduced；In view of the connexon of G tetramer higher structure is generally the ordered structures such as Tn or T+A, non-T base is changed For T；For T5G4T2G2T3G3 of the ordered structure such as since the 9th T, (" T5G4T2G2T3G3 " refers to continuous 5 herein T, 4 G etc.), only take continuous T to truncate, T sports the processing such as A；For the non-G base in first 8, substantially using poor Act method.

As shown in table 6,

D1 rite-directed mutagenesis adaptor molecules group includes: that 1A base is changed to C/T/G/X, 2A base and is changed to T/G and 1A2A alkali Base is changed to 7 sequences such as T base simultaneously；

D2 rite-directed mutagenesis adaptor molecules group includes: that 6C base is changed to T/A/G/X, 8G base and is changed to T base, 6C8G simultaneously It is changed to T base, 8 sequences such as 11T base is changed to X and 10T11T base while being changed to X；

D3&4 rite-directed mutagenesis adaptor molecules group includes: that 18T base is changed to A base and 23T base is changed to A base etc. 2 Sequence；

D5 rite-directed mutagenesis adaptor molecules group includes: that 27G base is changed to 3 sequences such as A/T/X；

GQ rite-directed mutagenesis adaptor molecules group includes: that 25G is changed to T/X (X represents nothing), i.e. 25T, 25X；It is changed in 6 C Under the premise of T, the G base in 14G, 15G, 16G, 17G is changed to T base, as 14T, 15T, 16T, 17T respectively, and Two G bases in 14G15G, 15G16G, 16G17G, 14G17G are changed to T base, i.e. 14T15T, 15T16T, 16T17T, 10 sequences such as 14T17T；

Degenerate sequence adaptor molecules group includes 1A6C base while being changed to T base, i.e. 1T6T；1A6C27G base difference It is changed to 1T6T27A base；And 1A27G base is changed to 3 sequences such as 1T27A base.

Above-mentioned aptamers stepping type combinatorial libraries are synthesized by Shanghai Sheng Gong bio-engineering corporation.

The rite-directed mutagenesis adaptor molecules group of table 6In27

Note: in table 6, "~" indicates the base deletion of the position.

Single site ac tive evaluation of the embodiment 14GQ rite-directed mutagenesis molecular group on SA chip

All sequences in GQ rite-directed mutagenesis adaptor molecules group, including such as SEQ ID NO.57~SEQ ID NO.66 institute In27-6T14T, In27-6T15T for showing, In27-6T16T, In27-6T17T, In27-6T1415TT, In27-6T1516TT, In27-6T1617TT, In27-6T1417T, In27-6T25X, In27-6T25T and reference sequence In27 (such as SEQ ID Shown in NO.33), totally 11 sequences, measuring the stock solutions of above-mentioned 11 sequences of 2 μ L respectively, (stock solution is configured with ultrapure water, dense Degree is 100 μM), 5 × Tris-HCl buffer of 4 μ L is added, 20,12 μ L of the 1wt%Tween sterilizing ultrapure water of 2 μ L obtains Stock solution among 10 μM of each sequence.Stock solution (10 μM) among each sequence of 20 μ L is taken, the Tris-T buffering of 380 μ L is added Liquid obtains each sequence operative solution of 500nM, 5min, cooled to room temperature is denaturalized at 95 DEG C.

Using securing NH₂The SA chip of-Bio-EPO- α, with the GQ rite-directed mutagenesis adaptor molecules group of the 500nM In each sequence and reference sequence In27 be analyte, using single-point screening method carry out SPR dynamic analysis.Specific experiment Condition is the same as embodiment 7.After sample introduction, analysans generates compound in conjunction with the EPO- α on SA chip, and sample introduction terminates, and injects Blank buffer solution, the compound on chip dissociate, each sequence single point R U value change curve such as Figure 11 A and Figure 11 B institute Show.

The result shows that being mutated compared with In27 to the G base on 14 to 17, response is all substantially reduced, Illustrate there may be certain mobility to the formation of GG skeleton in third.And In27-25X and In27-25T activity is also basic It loses, illustrates that 25G may take part in the 4th pair of GG skeleton and be formed.

Single site ac tive evaluation of the 15 D1 rite-directed mutagenesis adaptor molecules group of embodiment on SA chip

All sequences in D1 rite-directed mutagenesis adaptor molecules group, including such as SEQ ID NO.37~SEQ ID NO.43 institute In27-1X, In27-1T, In27-1G, In27-1C, In27-2G, In27-2T, the In27-1T2T and reference sequence shown In27,39nt, totally 9 sequences, measuring the stock solutions of above-mentioned 9 sequences of 2 μ L respectively, (stock solution is configured with ultrapure water, concentration It is 100 μM), 5 × Tris-HCl buffer of 4 μ L is added, 20,12 μ L of the 1wt%Tween sterilizing ultrapure water of 2 μ L obtains 10 μM each sequence among stock solution.Stock solution (10 μM) among each sequence of 20 μ L is taken, the Tris-T buffer of 380 μ L is added, Each sequence operative solution of 500nM is obtained, 5min, cooled to room temperature are denaturalized at 95 DEG C.

Using securing NH₂The SA chip of-Bio-EPO- α, with the D1 rite-directed mutagenesis adaptor molecules group of the 500nM In each sequence and reference sequence In27,39nt be analyte, using single-point screening method carry out SPR dynamic analysis.Specifically Experiment condition is the same as embodiment 7.Each sequence single point R U value change curve is as shown in figure 12.

The result shows that the response of In27-1T slightly increases, and the response of In27-1C and In27 are basic compared with In27 Maintain an equal level, response and the 39nt of In27-1G remains basically stable.And the response decline of In27-2G, In27-2T and In27-1T2T Obviously.Illustrate that 1A can be changed to other three kinds of bases without influencing activity in D1 structural domain, and 2A base has specificity.

Single site ac tive evaluation of the 16 D2 rite-directed mutagenesis adaptor molecules group of embodiment on SA chip

All sequences in D2 rite-directed mutagenesis adaptor molecules group, including such as SEQ ID NO.44~SEQ ID NO.51 institute In27-6X, In27-6T, In27-6A, In27-6G, In27-8T, In27-6T8T, In27-11X, the In27-10X11X shown, And reference sequence In27, totally 9 sequences, measure the stock solution (100 μM) of above-mentioned 9 sequences of 2 μ L respectively, and the 5 of 4 μ L are added × Tris-HCl buffer, 20,12 μ L of the 1wt%Tween sterilizing ultrapure water of 2 μ L, obtains deposit among 10 μM of each sequence Liquid.Stock solution (10 μM) among each sequence of 20 μ L is taken, the Tris-T buffer of 380 μ L is added, obtains each sequence of 500nM Working solution is denaturalized 5min, cooled to room temperature at 95 DEG C.

Using securing NH₂The SA chip of-Bio-EPO- α, in the D2 rite-directed mutagenesis adaptor molecules group of the 500nM Each sequence and reference sequence In27 are analyte, carry out SPR dynamic analysis using single-point screening method.Specific experiment condition is same Embodiment 7.Each sequence single point R U value change curve is as shown in figure 13.

The result shows that the affine activity of In27-6T (RU 60) is slightly higher compared with In27, and In27-6A (RU 30) and The response of In27-6G (RU 29) is declined.In27-6X (RU 15), In27-11X (RU 3) and In27-10X11X (RU 5) response decline is obvious and different from the dynamic characteristic of other sequences, and the rate of this three sequence association and dissociation is all It is very fast.To illustrate that D2 structural domain is important recognition site, the structure that 11 bases are formed ensure that identification activity Performance.

Single site ac tive evaluation of the embodiment 17D3&D4 rite-directed mutagenesis adaptor molecules group on SA chip

All sequences in D3&D4 rite-directed mutagenesis adaptor molecules group, including such as SEQ ID NO.52~SEQ ID In27-18A, In27-23A shown in NO.53 and reference sequence In27, totally 3 sequences, measure above-mentioned 3 sequences of 2 μ L respectively The stock solution (stock solution is configured with ultrapure water, and concentration is 100 μM) of column, is added 5 × Tris-HCl buffer of 4 μ L, 2 μ L's 20,12 μ L of 1wt%Tween sterilizing ultrapure water, obtains stock solution among 10 μM of each sequence.Take storage among each sequence of 20 μ L Standby liquid (10 μM) are added the Tris-T buffer of 380 μ L, obtain each sequence operative solution of 500nM, be denaturalized at 95 DEG C 5min, cooled to room temperature.

Using securing NH₂The SA chip of-Bio-EPO- α, with the D3&D4 rite-directed mutagenesis adaptor molecules of the 500nM Each sequence and reference sequence In27 in group are analyte, carry out SPR dynamic analysis using single-point screening method.Specific experiment Condition is the same as embodiment 7.Each sequence single point R U value change curve is as shown in figure 14.

The result shows that the response decline of In27-18A (RU 8) and In27-23A (RU 8) are obvious compared with In27. Illustrate that D3 and D4 structural domain plays certain effect in identification process.

Single site ac tive evaluation of the embodiment 18D5 rite-directed mutagenesis aptamers ergophore molecular group on SA chip

All sequences in D5 rite-directed mutagenesis adaptor molecules group, including such as SEQ ID NO.54~SEQ ID NO.56 institute In27-27X, In27-27A, the In27-27T and reference sequence In27 shown, totally 4 sequences, measure 2 μ L above-mentioned 4 respectively The stock solution (stock solution is configured with ultrapure water, and concentration is 100 μM) of sequence, is added 5 × Tris-HCl buffer of 4 μ L, 2 μ L 20,12 μ L of 1wt%Tween sterilize ultrapure water, obtain stock solution among 10 μM of each sequence.Among each sequence for taking 20 μ L Stock solution (10 μM) is added the Tris-T buffer of 380 μ L, obtains each sequence operative solution of 500nM, be denaturalized at 95 DEG C 5min, cooled to room temperature.

Using securing NH₂The SA chip of-Bio-EPO- α, in the D5 rite-directed mutagenesis adaptor molecules group of the 500nM Each sequence and reference sequence In27 be analyte, using single-point screening method carry out SPR dynamic analysis.Specific experiment condition With embodiment 7.Each sequence single point R U value change curve is as shown in figure 15.

The result shows that compared with In27, the sound of In27-27X (RU 6), In27-27T (RU 5) and In27-27A (RU 3) It is obvious that decline should be worth.Illustrate that 27G base has specificity.

Single site ac tive evaluation of the 19 degenerate sequence adaptor molecules group of embodiment on SA chip

All sequences in degenerate sequence adaptor molecules group, including as shown in SEQ ID NO.67~SEQ ID NO.69 In27-1T6T, In27-1T27A, In27-1T6T27A and reference sequence In27, totally 4 sequences, measure respectively on 2 μ L The stock solution (stock solution is configured with ultrapure water, and concentration is 100 μM) for stating 4 sequences, is added 5 × Tris-HCl buffering of 4 μ L Liquid, 20,12 μ L of the 1%Tween sterilizing ultrapure water of 2 μ L, obtains stock solution among 10 μM of each sequence.Take each sequence of 20 μ L Intermediate stock solution (10 μM) is added the Tris-T buffer of 380 μ L, obtains each sequence operative solution of 500nM, become at 95 DEG C Property 5min, cooled to room temperature.

Using securing NH₂The SA chip of-Bio-EPO- α, in the degenerate sequence adaptor molecules group of the 500nM Each sequence and reference sequence In27 are analyte, carry out SPR dynamic analysis using single-point screening method.Specific experiment condition is same Embodiment 7.Each sequence single point R U value change curve is as shown in figure 16.

The result shows that compared with In27, the response of 1T6T is significantly raised, at the same the response of 1T6T between 1T and 6T it Between.The decline of 1T27A and 1T27A6T response is obvious, illustrates that 27 bit bases (G) have played important work in identification process again With, and it is unrelated with the pairing of both ends 1T27A.

Embodiment 20 carries out affine recognition detection using In27 as recognition component, with EPO- α

Take the 3 '-dT of 2 μ L₁₂(by Shanghai, Sheng Gong bioengineering Co., Ltd synthesizes-Biotin-In27, at the 3 ' ends of In27 Modify a Biotin molecule) stock solution (stock solution is configured with ultrapure water, and concentration is 100 μM), 5 × Tris-HCl is added 20,26 μ L of the 1wt%Tween sterilizing ultrapure water of buffer 8 μ L, 4 μ L, obtain 5 μM of 3 '-dT₁₂Storage among-Biotin-In27 Standby liquid.The intermediate stock solution (5 μM) of 4 μ L is taken, the Tris-T buffer of 396 μ L is added, the fixed solution of 50nM is obtained, in 95 DEG C Lower denaturation 5min, 4 DEG C of placements.

According to the above method, the 3 '-Biotin-39nt of the 50nM of 400 μ L are configured (by Shanghai Sheng Gong bioengineering Co., Ltd Synthesis, in a 3 ' terminal modified upper Biotin molecules of 39nt) fixed solution.

One piece of new SA chip is taken, fixed pre-treatment is the same as embodiment 5.The 3 '-Biotin-39nt fixation of 50nM is molten Liquid is fixed in SA chip channel 2, at this time response stability under conditions of sample injection time 300 seconds in 10 μ L/min of flow velocity (stability) RU value rises 1550.By the 3 '-dT of 50nM₁₂- Biotin-In27 fixed solution, in 10 μ L/min of flow velocity, It under conditions of sample injection time 300 seconds, is fixed in SA chip channel 4, response stability (stability) RU value rises at this time 1800。

On SA chip, 3 '-Biotin-39nt (channel 2) and 3 '-dT are used₁₂- Biotin-In27 (channel 4) is carried out Affinity and dynamic analysis between EPO- α and 39nt and In27.Binding time is 120 seconds, and Dissociation time is 300 seconds, flow velocity For 30 μ L/min.Regeneration condition is 5mM NaOH, and sample injection time 15 seconds, flow velocity was 30 μ L/min.Comprising being zero with blank solvent The Loading sequence of six concentration of EPO- α including point is 0,0,6.25,12.5,25,50,100,200nM, obtains going against accepted conventions in conjunction with solution Figure, as shown in Figure 17 A and Figure 17 B.

Nonlinear fitting the result shows that, 39nt and EPO- α interaction dissociation constant K_D=55nM, association rate constant K_A=2.7×10⁴M^-1s^-1, dissociation rate constant K_d=1.4 × 10^-4s^-1(U-value=1, Chi²=0.23)；In27 and EPO- α The dissociation constant K of interaction_D=78nM, association rate constant K_a=2.2 × 10⁴M^-1s^-1, dissociation rate constant K_d=1.7 × 10^-4s^-1(U-value=1, Chi²=0.06).Illustrate that In 27 as recognition component, is realized well to EPO- α albumen Affine identification and detection.

SEQUENCE LISTING

<110>Inst of Toxic Medicinal Materials, P.L.A. Academy of Military Medical Sciences

<120>a kind of aptamers combinatorial libraries, its construction method and purposes

<130> IDC160136

<160> 69

<170> PatentIn version 3.5

<210> 1

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agagagagag agagaaaggt ctgtttttgg ggttggtttg ggtctctctc tctctct 57

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tgtttttggg gttggtttgg gtcaata 27

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tttttggggt tggtttgggt caata 25

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tttggggttg gtttgggtca ata 23

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tggggttggt ttgggtcaat a 21

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attgaaaggt ctgtttttgg ggttggtttg ggtcaat 37

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ttgaaaggtc tgtttttggg gttggtttgg gtcaa 35

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tgaaaggtct gtttttgggg ttggtttggg tca 33

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gaaaggtctg tttttggggt tggtttgggt c 31

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aaaggtctgt ttttggggtt ggtttgggt 29

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aaggtctgtt tttggggttg gtttggg 27

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aggtctgttt ttggggttgg tttgg 25

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ggtctgtttt tggggttggt ttg 23

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gtctgttttt ggggttggtt t 21

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aggtctgttt ttggggttgg tttggg 26

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taggtctgtt tttggggttg gtttggg 27

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gaggtctgtt tttggggttg gtttggg 27

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caggtctgtt tttggggttg gtttggg 27

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agggtctgtt tttggggttg gtttggg 27

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atggtctgtt tttggggttg gtttggg 27

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ttggtctgtt tttggggttg gtttggg 27

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aaggttgttt ttggggttgg tttggg 26

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aaggtttgtt tttggggttg gtttggg 27

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aaggtatgtt tttggggttg gtttggg 27

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aaggtgtgtt tttggggttg gtttggg 27

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aaggtctttt tttggggttg gtttggg 27

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aaggtttttt tttggggttg gtttggg 27

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aaggtctgtt ttggggttgg tttggg 26

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aaggtctgtt tggggttggt ttggg 25

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aaggtctgtt tttggggatg gtttggg 27

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aaggtctgtt tttggggttg gtatggg 27

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aaggtctgtt tttggggttg gtttgg 26

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aaggtctgtt tttggggttg gtttgga 27

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aaggtctgtt tttggggttg gtttggt 27

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aaggtttgtt ttttgggttg gtttggg 27

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aaggtttgtt tttgtggttg gtttggg 27

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aaggtttgtt tttggtgttg gtttggg 27

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aaggtttgtt tttgggtttg gtttggg 27

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aaggtttgtt tttttggttg gtttggg 27

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aaggtttgtt tttgttgttg gtttggg 27

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aaggtttgtt tttggttttg gtttggg 27

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aaggtttgtt ttttggtttg gtttggg 27

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aaggtttgtt tttggggttg gtttgg 26

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aaggtttgtt tttggggttg gttttgg 27

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taggtttgtt tttggggttg gtttggg 27

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taggtctgtt tttggggttg gtttgga 27

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taggtttgtt tttggggttg gtttgga 27

Claims (39)

1. a kind of method for constructing aptamers combinatorial libraries, comprising the following steps:

The aptamers to be optimized with N number of base are provided, using aptamers to be optimized as initiation sequence；

The first molecular group is constructed, it includes n₁A molecule, the n₁A molecule is respectively 5 ' end truncates of initiation sequence, and And each molecule relative to initiation sequence there are 5 ' different ends to truncate length；

The second molecular group is constructed, it includes n₂A molecule, the n₂A molecule is respectively 3 ' end truncates of initiation sequence, and And each molecule relative to initiation sequence there are 3 ' different ends to truncate length；

Third molecular group is constructed, it includes n₃A molecule, the n₃A molecule is respectively 5 ' and 3 ' ends of initiation sequence while cutting Short truncate, 5 ' is identical with 3 ' ends truncation length in a molecule, and each molecule has relative to initiation sequence There is different ends to truncate length；

Optionally, the 4th molecular group is constructed, it includes n₄A molecule,

When initiation sequence is capable of forming loop-stem structure, the n₄A molecule is respectively the ring portion Series extension body of initiation sequence, institute Stem's sequence that ring portion Series extension body does not include initiation sequence is stated, includes 1~n in 5 ' ends₄A First ray, and at 3 ' ends End includes 1~n₄A second sequence, second sequence are the reverse complementary sequences of First ray, the 5 ' ends in a molecule The second sequence quantity that the First ray for including and 3 ' ends include is identical, and each molecule has relative to initiation sequence Different terminal extension length；

When initiation sequence cannot form loop-stem structure, the n₄A molecule is respectively the extension body of initiation sequence, the extension body 5 ' ends include 1~n₄A First ray, and include 1~n in 3 ' ends₄A second sequence, second sequence are the first sequence The reverse complementary sequence of column, the second sequence quantity phase that the First ray and 3 ' ends that 5 ' ends include in a molecule include Together, and each molecule relative to initiation sequence have different terminal extension length,

The length of the First ray is one or more bases,

Wherein, N is the integer greater than zero,

n₁、n₂、n₃、n₄It is each independently greater than zero and is less than or equal to the integer of N.

2. the method for building aptamers combinatorial libraries described in claim 1, the sequence that wherein First ray is made of A and G, The sequence that second sequence is made of C and T.

3. it is described in claim 1 building aptamers combinatorial libraries method, wherein the length of the First ray be 1,2,3 or 4 bases.

4. the method for building aptamers combinatorial libraries described in claim 1, wherein N is the integer between 30~130.

5. the method for building aptamers combinatorial libraries described in claim 1, wherein n₁、n₂、n₃、n₄It is each independently 5~80 Between integer.

6. the method for building aptamers combinatorial libraries described in claim 1, wherein

The construction method of first molecular group are as follows: initiation sequence is truncated from 5 ' ends, carries out n altogether₁It is secondary, every time truncate 1 or Multiple bases, thus to obtain n₁A truncated molecule in 5 ' end constitutes the first molecular group；

The construction method of second molecular group are as follows: initiation sequence is truncated from 3 ' ends, carries out n altogether₂It is secondary, every time truncate 1 or Multiple bases, thus to obtain n₂A truncated molecule in 3 ' end constitutes the second molecular group；

The construction method of third molecular group are as follows: initiation sequence is truncated simultaneously from 5 ' and 3 ' ends, carries out n altogether₃It is secondary, every time One or more bases are truncated simultaneously to 5 ' and 3 ' ends, thus to obtain n₃A 5 ' and 3 ' ends truncated molecule simultaneously, constitutes the Three molecular groups；

The construction method of 4th molecular group are as follows:

When initiation sequence is capable of forming loop-stem structure, keeps the ring portion sequence of initiation sequence constant, stem's sequence is removed, First ray is added in 5 ' ends of ring portion sequence, and adds the second sequence in 3 ' ends of ring portion sequence, and second sequence is The reverse complementary sequence of First ray, repeats n₄It is secondary, thus to obtain n₄A 5 ' and 3 ' ends while extended molecule are constituted 4th molecular group；

When initiation sequence cannot form loop-stem structure, First ray is added in 5 ' ends of initiation sequence, and add in 3 ' ends Add the second sequence, second sequence is the reverse complementary sequence of First ray, repeats n₄It is secondary, thus to obtain n₄A 5 ' and 3 ' ends while extended molecule constitute the 4th molecular group.

7. the method for building aptamers combinatorial libraries as claimed in claim 6, in which:

When constructing the first molecular group, 1,2,3 or 4 base is truncated every time；

When constructing the second molecular group, 1,2,3 or 4 base is truncated every time；

When constructing third molecular group, 1,2,3 or 4 base is truncated simultaneously to 5 ' and 3 ' ends every time.

8. the method for building aptamers combinatorial libraries described in claim 1, wherein the first molecular group, the second molecular group or third The shortest molecule of length in molecular group has 10~40 bases.

9. the method for building aptamers combinatorial libraries according to any one of claims 8, wherein the first molecular group, the second molecular group or third The shortest molecule of length in molecular group has 20~30 bases.

10. the method for the described in any item building aptamers combinatorial libraries of claim 1 to 9, wherein the aptamers to be optimized For the aptamers for specifically binding epo protein.

11. the method for building aptamers combinatorial libraries described in any one of claim 10, wherein the aptamers to be optimized are SEQ ID Overall length aptamers shown in NO.1.

12. a kind of aptamers combinatorial libraries, comprising:

First molecular group, it includes n₁A molecule, the n₁A molecule is respectively 5 ' end truncates of initiation sequence, and every One molecule relative to initiation sequence there are 5 ' different ends to truncate length；

Second molecular group, it includes n₂A molecule, the n₂A molecule is respectively 3 ' end truncates of initiation sequence, and every One molecule relative to initiation sequence there are 3 ' different ends to truncate length；

Third molecular group, it includes n₃A molecule, the n₃5 ' and 3 ' ends that a molecule is respectively initiation sequence are truncated simultaneously Truncate, 5 ' is identical with 3 ' ends truncation length in a molecule, and each molecule has not relative to initiation sequence Same end truncates length；And

Optionally, the 4th molecular group, it includes n₄A molecule,

When initiation sequence is capable of forming loop-stem structure, the n₄A molecule is respectively the ring portion Series extension body of initiation sequence, institute Stem's sequence that ring portion Series extension body does not include initiation sequence is stated, includes 1~n in 5 ' ends₄A First ray, and at 3 ' ends End includes 1~n₄A second sequence, second sequence are the reverse complementary sequences of First ray, the 5 ' ends in a molecule The second sequence quantity that the First ray for including and 3 ' ends include is identical, and each molecule has relative to initiation sequence Different terminal extension length；

When initiation sequence cannot form loop-stem structure, the n₄A molecule is respectively the extension body of initiation sequence, the extension body 5 ' ends include 1~n₄A First ray, and include 1~n in 3 ' ends₄A second sequence, second sequence are the first sequence The reverse complementary sequence of column, the second sequence quantity phase that the First ray and 3 ' ends that 5 ' ends include in a molecule include Together, and each molecule relative to initiation sequence have different terminal extension length,

The length of the First ray is one or more bases；

Wherein, N is the integer greater than zero,

n₁、n₂、n₃、n₄It is each independently greater than zero and is less than or equal to the integer of N,

The initiation sequence is aptamers to be optimized.

13. aptamers combinatorial libraries described in claim 12, the sequence that wherein First ray is made of A and G, the second sequence The sequence being made of C and T.

14. aptamers combinatorial libraries described in claim 12, wherein the length of the First ray is 1,2,3 or 4 base.

15. aptamers combinatorial libraries described in claim 12, wherein N is the integer between 30~130.

16. aptamers combinatorial libraries described in claim 12, wherein n₁、n₂、n₃、n₄It is each independently whole between 5~80 Number.

17. aptamers combinatorial libraries described in claim 12, wherein the aptamers to be optimized are specific binding epo protein Aptamers.

18. aptamers combinatorial libraries described in claim 17, wherein the aptamers to be optimized are shown in SEQ ID NO.1 Overall length aptamers.

19. aptamers combinatorial libraries described in claim 17 comprising: sequence shown in SEQ ID NO.2~SEQ ID NO.36 Column.

20. a kind of method for screening aptamers comprising: text is combined using the described in any item aptamers of claim 12 to 19 Aptamers needed for library is screened.

21. the method for screening aptamers described in claim 20, wherein required aptamers are that can specifically bind epo protein Aptamers.

22. the method for screening aptamers described in claim 20 comprising:

(1) aptamers combinatorial libraries are provided；

(2) each of aptamers combinatorial libraries molecule is contacted with target protein；

(3) affinity in aptamers combinatorial libraries between each molecule and target protein is detected.

23. the method for screening aptamers described in claim 22, wherein the target protein is epo protein.

24. the method for screening aptamers described in claim 22, this method is examined using surface plasmon resonance (SPR method) Survey the affinity between each molecule and target protein.

25. the method for screening aptamers described in claim 22, comprising the following steps:

1) target protein is fixed on chip surface；

2) SPR dynamic analysis is carried out, sample introduction makes the molecule in the aptamers combinatorial libraries and is fixed on the target of chip surface Protein binding generates compound, and sample introduction terminates, and injects blank buffer solution, and the compound on chip dissociates, and obtains each sequence List point RU value change curve, judges whether aptamers to be screened are active according to RU value, and aptamers of the RU value greater than 0 have Activity；

3) it for active molecule, carries out multi-cycle dynamic analysis (MCK analysis), obtains dissociation constant K_D, according to K_DNumber The size of value judges the affinity between each molecule and target protein, K_DNumerical value is smaller, and affinity is stronger.

26. the method for screening aptamers described in claim 25, wherein the chip is SA chip.

27. the method for screening aptamers described in claim 25, wherein be fixed on chip after the target protein couple biotin Surface.

28. the method for screening aptamers described in claim 27, wherein the target protein is at the amino end of amino acid residue The sialic acid terminal and biotin covalent coupling at end, carboxyl terminal or glycosyl.

29. the method for screening aptamers described in claim 27 or 28, wherein the chemistry of target protein and biotin covalent coupling Metering is than being 1:1.

30. the method for screening aptamers described in claim 25, wherein the molecule in step 2) the aptamers combinatorial libraries In 90~98 DEG C of 5~10min of denaturation, cooled to room temperature, then sample introduction, injector temperature are 20~30 DEG C, sample introduction binding time It is 60~180 seconds, Dissociation time is 120~600 seconds, and flow velocity is 20~50 μ L/min, and buffer used is Tris-T buffer.

31. the method for screening aptamers described in claim 30, wherein step 2) have selected from it is following i) into vii) one A or multiple features:

I) denaturation temperature is 95 DEG C；

Iii) denaturation time is 5min；

Iv) injector temperature is 25 DEG C；

V) sample introduction binding time is 120 seconds；

Vi) Dissociation time is 300 seconds；

Vii) flow velocity is 30 μ L/min.

32. the method for screening aptamers described in claim 25, wherein the analysis of MCK described in step 3), by the aptamers For molecule in combinatorial libraries in 90~98 DEG C of 5~10min of denaturation, cooled to room temperature, then sample introduction, injector temperature is 20~30 DEG C, sample introduction binding time is 60~180 seconds, and Dissociation time is 120~600 seconds, and flow velocity is 20~50 μ L/min, and MCK was analyzed Buffer used is Tris-T buffer in journey.

33. the method for screening aptamers described in claim 32, wherein the analysis of MCK described in step 3) have selected from it is following i) One or more features into vii):

I) denaturation temperature is 95 DEG C；

Iii) denaturation time is 5min；

Iv) injector temperature is 25 DEG C；

V) sample introduction binding time is 120 seconds；

Vi) Dissociation time is 300 seconds；

Vii) flow velocity is 30 μ L/min.

34. aptamers, the sequence shown in NO.5~9 SEQ ID, 33,38~40,45 and 67.

35. aptamers described in claim 34 are preparing the purposes in kit or chip for detecting epo protein.

36. purposes described in claim 35, wherein the epo protein be selected from natural EPO albumen, rHuEPO- α albumen, RHuEP O- β albumen and deglycosylated above-mentioned epo protein.

37. composition, kit or chip for detecting epo protein, wherein containing the aptamers described in claim 34.

38. for detecting the composition, kit or chip of epo protein described in claim 37, wherein the epo protein Selected from natural EPO albumen, rHuEPO- α albumen, rHuEP O- β albumen and deglycosylated above-mentioned epo protein.

39. the method for the detection epo protein of non-disease diagnostic purpose, comprising:

(1) sample to be tested is provided；

(2) aptamers described in claim 34 are contacted with sample to be tested；

(3) combination between aptamers and EPO is detected.