CN109868268A - A method of optimization DNA encoding library of compounds Start Fragment - Google Patents
A method of optimization DNA encoding library of compounds Start Fragment Download PDFInfo
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- CN109868268A CN109868268A CN201711263372.3A CN201711263372A CN109868268A CN 109868268 A CN109868268 A CN 109868268A CN 201711263372 A CN201711263372 A CN 201711263372A CN 109868268 A CN109868268 A CN 109868268A
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- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229940029575 guanosine Drugs 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 125000005647 linker group Chemical group 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 125000002270 phosphoric acid ester group Chemical group 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 229940079827 sodium hydrogen sulfite Drugs 0.000 description 1
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- TUQOTMZNTHZOKS-UHFFFAOYSA-N tributylphosphine Chemical compound CCCCP(CCCC)CCCC TUQOTMZNTHZOKS-UHFFFAOYSA-N 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
Abstract
The invention discloses a kind of methods for optimizing DNA encoding library of compounds Start Fragment, wherein there is the site that can be cut off by mild condition shirtsleeve operation in Start Fragment.Optimize DNA encoding library of compounds Start Fragment as obtained by this method, and method mild condition, easy to operate, safety and environmental protection, segment yield are higher.
Description
Technical field
The present invention relates to a kind of methods for optimizing DNA encoding library of compounds Start Fragment.
Background technique
During new drug development, in spite of very more challenges, but the most fundamental still it is new drug guide's chemical combination
The discovery and optimization of object, wherein for biological target high flux screening be quickly obtained lead compound main means it
One.In order to quickly search out the lead compound of excellent, establishing large-scale library of molecules is just particularly important, this
The foundation in one library of sample is the starting point and support of drug target screening.
Traditional high flux screening relies on the library of molecules of substantial amounts, tentatively realizes the scale of drug screening, larger
Medical substance resource is utilized to limit, improves the probability of drug discovery.However, its library of molecules limited amount, at present only
Million grades of compounds.And the foundation in such a library needs to synthesize or buy for a long time, it usually needs 10 years or more products
Tired, price is very expensive, and the maintenance of library of molecules is also extremely complex.The drug screening period is long, exists in practical applications huge
Barrier.
The DNA encoding compound library synthetic technology occurred in recent years, combines combinatorial chemistry and Protocols in Molecular Biology,
Each compound is added into a DNA label on molecular level, up to hundred million grades of compound library can be synthesized in a very short period of time.
And compound can be identified by the method for gene sequencing, be substantially increased the size and synthesis effect of compound library
Rate becomes the trend of next-generation compound library screening technique, and starts to be widely applied in external pharmaceutical industry, produces many products
The effect of pole.Although combinatorial chemistry synthetic method can quickly generate huge number of compound mixture, in screening process
It can not learn the compound information to work.Therefore, be born DNA encoding library of compounds technology on this basis.This is close
The relatively new and faster molecular level compound label technology of development over year.
Its implementation process is on the basis of conventional combination chemistry, by a specific compound and one section of unique sequences
DNA is linked in molecular level, i.e., carries out DNA encoding to small molecule compound.There are one with DNA sequence dna for the structural unit of compound
One corresponding relationship.After carrying out affine screening with respective target point, so that it may by the identification to DNA sequence dna to identify compound
Structural information, then the compound being translated is synthesized, active confirmation is carried out with target, to obtain guide's chemical combination
Object.Solve the problems, such as that the huge compound library that combinatorial chemistry generates is not used to lead compound screening.
Huge DNA encoding library of compounds synthesis changes the pattern of lead compound discovery with the appearance of screening technique.
Compared with million grades of compound libraries of traditional high flux screening, DNA encoding library of compounds is in compound amounts (tens or more
It is more) and multifarious covering on be all increased considerably.Moreover, the type library in a relatively short period of time can be built up,
Usually only need one month;The maintenance of library of molecules is extremely simple, and -80 DEG C of refrigerators can store hundreds and thousands of similar texts
Library.The R&D cycle is substantially reduced, research and development cost is reduced.
Under normal conditions, double-stranded DNA is stablized compared with single stranded DNA, can be compatible with the chemical reaction of DNA also relative abundance, being capable of structure
More huge, multifarious library is built, therefore people preferentially use double stranded oligonucleotide to carry out compound structural unity
Label.Patent application publication number US5573905 describes the tripeptide compound using single strain oligonucleotide label 2 × 2 × 2
The theory in library opens the chapter of DNA encoding library of compounds.Patent application publication number US0158765 discloses a kind of benefit
With the method in double stranded oligonucleotide labeled compound library, double stranded oligonucleotide Start Fragment passes through long-chain attachment phase
Even.Patent application publication number US0053091 discloses a kind of method using double stranded oligonucleotide labeled compound library,
Its Start Fragment is the hairpin structure with derivative nonnatural base.Patent application publication number WO127933, describes and is based on
The method for building up of oligonucleotide templates library of compounds is another entirely different double-stranded DNA coding techniques.
But library of compounds is encoded using double-stranded DNA, there is also many deficiencies: for example to library of compounds
When carrying out affinity screening, need in advance to carry out target point protein immobilized;The hair that DNA encoding library of compounds Start Fragment has
Card structure or non-natural nucleic acid sequence can reduce subsequent PCR amplification efficiency, or even cause mispairing, bring for DNA sequencing
It is difficult.
It is, thus, sought for a kind of mild condition, easy to operate, safety and environmental protection, yield is higher and is easy to DNA sequencing
The optimization method of DNA encoding compound library, to promote extensive exploitation and application of such method in field of medicaments.
Summary of the invention
In order to solve described problem, inventor provides a kind of condition temperature aiming at the problem that PCR amplification efficiency in the prior art
With the method for the higher optimization DNA encoding library of compounds Start Fragment of easy to operate, safety and environmental protection, yield, to promote such
Extensive exploitation and application of the method in field of medicaments.
The present invention provides a kind of methods for optimizing DNA encoding library of compounds Start Fragment.
To realize the goal of the invention, The technical solution adopted by the invention is as follows: a kind of optimization DNA encoding library of compounds
The method of Start Fragment is the double-stranded DNA for the DNA encoding library of compounds Start Fragment or any circulation for making to have Formulas I structure,
By mild condition shirtsleeve operation to wherein a chain carries out selective rhizotomy or two chains are cut off simultaneously, do not damaging
Single stranded DNA is obtained in the case where library and encoded information, has and shows as follows:
The method that the present invention optimizes DNA encoding library of compounds Start Fragment, includes the following steps:
A) one Start Fragment is provided, wherein the funtion part of the Start Fragment includes one or more reactive group R, and
And another part of Start Fragment connects oligonucleotide ssDNA;
B) funtion part of the Start Fragment and the structural unit containing at least one reaction site are covalently attached,
The reactive group R of the reaction site of middle compound structural unity and Start Fragment funtion part is to be covalently attached;
C) on the oligonucleotide ssDNA of the starting of the Start Fragment, the structure list of markers step b) is capable of in introducing
Other coding oligonucleotides of member;
Wherein the structure I of the Start Fragment is as follows:
Wherein,
R is the reactive group of Start Fragment funtion part;
A is connection atom or skeleton structure;
S is the gasket for connecting R and A;
SsDNA is the oligonucleotide in step a);
B1、B2、B3、B4It is suitable for the functional group of A and oligonucleotide bonding;
L1, L2 are the attachments for connecting A and oligonucleotide ssDNA;
X is the site that can be cut off by mild condition shirtsleeve operation.
In one embodiment, wherein the method also includes following steps:
D) DNA encoding library of compounds is combined with target albumen, collection can be with the library constructs of targeted integration;
E) coding oligonucleotide corresponding to the library constructs of step d) carries out PCR amplification and sequencing, determines energy and mesh
The structure for the library constructs that mark target point protein combines;
F) it is detached from DNA, and chemical synthesis is carried out to the library constructs that step e) is obtained, verifies its target validity.
In one embodiment, it step b) and c) can sequentially exchange.
In one embodiment, step a) forms n times circulation to n times c) are repeated, and wherein n is 2 or more integer, excellent
N is selected to be equal to 2 or 3 or 4.
In one embodiment, in step a) Start Fragment funtion part reactive group R be selected from amino, hydroxyl, sulfydryl,
Carboxyl, nitrine, Terminal Acetylenes, alkynes, tetrazine, the trans- interior alkene of octatomic ring, aldehyde, ketone, halogenated aromatic ring or heteroaromatic, alpha-halogenate in octatomic ring
Ketone or phosphorus ylide, the reactive group R of the Start Fragment funtion part are preferably amino or carboxyl.
In one embodiment, the reaction site of structural unit is with the reactive group R of Start Fragment funtion part with covalent
Connection, the reaction site of the structural unit be selected from amino, carboxyl, sulfonyl, halogenated aromatic ring, heteroaromatic, isocyanate group,
Aldehyde, ketone or alpha, beta-unsaturated ketone or ester.
In one embodiment, the reaction site of structural unit is with the reactive group R of Start Fragment funtion part with covalent
The reaction site of connection, the structural unit is selected from phosphorus ylide, aldehyde or ketone.
In one embodiment, the reaction site of structural unit is with the reactive group R of Start Fragment funtion part with covalent
Connection, the reaction site of the structural unit are selected from alkynes, tetrazine or the trans- interior alkene of octatomic ring in nitrine and Terminal Acetylenes, octatomic ring.
In one embodiment, the oligonucleotide ssDNA in step a) is single-stranded, and upper chain and the lower chain base sequence
Column complementary pairing, while thering is pairing of the prominent base sequence for next code oligonucleotide to identify.
In one embodiment, the oligonucleotide of the starting in step c) and other described coding oligonucleotides
It connects or is connected chemically by enzyme.
In one embodiment, the reactive group R of the Start Fragment funtion part not with A, B in Start Fragment and widow
Poly-nuclear glycosides ssDNA reaction.
In one embodiment, wherein the A is selected from-CH- or phloroglucin.
In one embodiment, the S is selected from oligoethylene glycol, lower alkylene, oligomeric N- methyl-D-alanine, low
Poly- N- methyl-L-alanine or oligomeric N- methyl DL-alanine.
In a specific embodiment, S preferably is selected from 1~20 polyethylene glycol or 1~20 poly- N- methylalanine.
In one embodiment, the B1Selected from phosphate, ester group, amido bond, carbamate, carbonate group or three
Nitrogen azoles, preferably phosphate;B2Selected from phosphate, ester group, amido bond, carbamate, carbonate group or triazole, preferably
For phosphate;B3Selected from phosphate, ester group, amido bond, carbamate, carbonate group or triazole, preferably phosphate;B4
Selected from phosphate, ester group, amido bond, carbamate, carbonate group or triazole, preferably phosphate.
In one embodiment, L1 is selected from oligoethylene glycol, lower alkylene, oligomeric N- methyl-D-alanine, oligomeric N-
Methyl-L-alanine or oligomeric N- methyl DL-alanine;L2 is selected from oligoethylene glycol, lower alkylene, oligomeric N- methyl-
D-alanine, oligomeric N- methyl-L-alanine or oligomeric N- methyl DL-alanine.
In a specific embodiment, L1 is 1~20 polyethylene glycol or 1~20 poly- N- methylalanine;L2 is 1~20 poly-
Ethylene glycol or 1~20 poly- N- methylalanine.
In one embodiment, the X is that can be digested disconnected functional group, and the functional group is selected from disulfide bond.
In a specific embodiment, the enzyme for cutting off the disulfide bond is thiol reduction enzyme.
In one embodiment, the X is the functional group that can be cut off by chemical reaction.
In one embodiment, the X is the functional group that can be cut off by nucleopilic reagent or alkali, the structure choosing of the functional group
Freely
Composed group.
In one embodiment, the X is selected from the functional group of silicon atoms, the structure of the functional group be selected from by
Composed group.
In a specific embodiment, the reagent for cutting off the functional group of the silicon atoms is selected from fluorine anion, preferably
Tetrabutyl ammonium fluoride or hydrogen fluoride pyridine.
In one embodiment, the X functional groups that can be reduced reagent cutting, the structure of the functional group be selected from by
Composed group.
In a specific embodiment, cut off the hydrazine key reagent be selected from samarium diodide, zinc/acetic acid, zinc/ethyl alcohol, iron/
Acetic acid, palladium carbon/hydrogen, Raney's nickel/hydrogen or indium/ethyl alcohol.
In a specific embodiment, the reagent for cutting off the azo bond is selected from sodium dithionite, sodium hydrogensulfite, palladium
Carbon/hydrogen, Raney's nickel/hydrogen, zinc/acetic acid or zinc/ethyl alcohol.
In a specific embodiment, cut off the nitrogen-oxygen bond reagent be selected from samarium diodide, zinc/acetic acid, zinc/ethyl alcohol,
Iron/acetic acid, palladium carbon/hydrogen, Raney's nickel/hydrogen or indium/ethyl alcohol.
In a specific embodiment, the reagent for cutting off the disulfide bond is selected from DTT, TCEP, triphenylphosphine, tributyl
Phosphine, 2 mercapto ethanol, 1,3- dimercaptopropane, zinc/acetic acid or triphenylphosphine radium chloride/hydrogen.
In a specific embodiment, when the X is selected from disulfide bond, the oligonucleotide of the starting in step c) with
Other described coding oligonucleotides are by being connected chemically.
In one embodiment, the described X functional group that can be cut off by organometallic reagent, the structure choosing of the functional group
Freely
Composed group.
In one embodiment, cut off the allyl or benzyl reagent be selected from four triphenyl phosphorus palladiums/triethylsilane,
Or palladium carbon/hydrogen.
In one embodiment, the X is the functional group that can be oxidized cutting, the structure of the functional group be selected from by
Composed group.
In a specific embodiment, the reagent for cutting off the vicinal diamines is sodium metaperiodate.
In a specific embodiment, cutting off the β-seleno compound reagent is hydrogen peroxide or elemental iodine.
In one embodiment, the X is selected from the functional group that can be cut off by weak acid, the structure of the functional group be selected from by
Composed group.
In a specific embodiment, the reagent for cutting off the functional group is acetic acid or magnesium chloride.
In one embodiment, the X is selected from the functional group that can be cut off by light reaction, the structure of the functional group be selected from by
Composed group.
In a specific embodiment, the optical wavelength range for cutting off the functional group is selected from 254-532nm.
In one embodiment, L1, L2 can be simultaneously containing cut-off site X.
In one embodiment, X can L1 or L2 any one.
Compared with prior art, the present invention have it is following significant the utility model has the advantages that
1, when establishing of DNA encoding library of compounds is being carried out, is using double-stranded DNA as encoded information, double-stranded DNA is utilized
Stability, the chemical reaction of DNA also relative abundance can be compatible with, can more construct more huge, multifarious library of compounds.
2, chain cutting condition agents useful for same is simple and easy to get, low in cost, commercially available, and is not related to poisonous and harmful danger
Dangerous product reagent, safety and environmental protection, simultaneous reactions mild condition, stable system is reproducible, and production cost is low, and high income can promote
Extensive exploitation and application of such method in field of medicaments.
3, before Sites Screening, selective rhizotomy is carried out to a wherein chain by mild condition shirtsleeve operation,
The coded compounds library with double-stranded DNA and the coded compounds library with single stranded DNA can be screened respectively.
4, the advantage and disadvantage unrestricted choice of two kinds of filtering modes, target spot especially unstable in face of structure, property can be directed to
Albumen improves operating flexibility.
5, before to DNA sequencing, selective rhizotomy, energy are carried out to a wherein chain by mild condition shirtsleeve operation
Enough substantially reducing hairpin structure bring in Start Fragment influences.By the transformation of double-stranded DNA to single stranded DNA, PCR can be greatly promoted
Amplification efficiency reduces the risk of base-pair mismatch, promotes the accuracy rate of DNA sequencing, so that entire coding techniques becomes more reliable.
Detailed description of the invention
Fig. 1 shows the mass-spectrogram of Start Fragment.
Fig. 2 shows two circular linkages to react Ago-Gel figure.
Fig. 3 is shown deconvolute before DNA encoding library of compounds second circulation synthesis mass-spectrogram.
Fig. 4 is second circulation synthesis mass-spectrogram of DNA encoding library of compounds after deconvoluting.
Fig. 5 is the base sequence PCR efficiency standard curve before being acted on using TCEP.
Fig. 6 is the base sequence PCR efficiency standard curve after being acted on using TCEP.
Specific embodiment
Below by specific embodiment and experimental data, the present invention is further illustrated.Although for clear mesh
, proprietary term is used below, but these terms are not meant to define or limit the scope of the invention.
As described herein, term " compound structural unity ", " structural unit " are interchangeable, refer to and may connect to other changes
It learns the unit of structure or can connect unit as other.When funtion part is polymer or oligomer, the knot
Structure unit is the monomeric unit of poly or oligomer.Structural unit can also include skeleton structure unit, can be connected to
One or more other structures (for example, peripheral structure unit).Structural unit can be complementary any compound (that is, knot
Structure unit allows for one and reacts).It, can also be in general, all structural units used can have at least two active groups
Allow some structural units that respectively only there is an active group.
As described herein, term " other coding oligonucleotide " refer to it is on Start Fragment other than oligonucleotide,
The nucleotide multimer of subsequent introducing, it is therefore an objective to which corresponding compound structural unity is marked.Wherein oligonucleotide can
To include DNA or its any derivative known in the art, being synthesized and be identified for base-pair.Oligonucleotide multimer
Body may include natural nucleus glycoside (for example, adenosine, thymidine, guanosine, cytidine, uridine, desoxyadenossine, deoxythymidine, deoxyguanosine with
And deoxycytidine), nucleoside analog, chemical modification base, bio-modification base, insertion base, modification sugar or/and modification phosphoric acid
Ester group.
Experimental method in following embodiments is unless otherwise specified conventional method.
Embodiment 1: the preparation of Start Fragment (S-HP)
The nucleotide chain with following sequence of 5 one-tenth phosphorylations has been synthesized as Start Fragment, and has passed through Suzhou Jin Weizhi
Biotechnology Co., Ltd carries out HPLC purifying.
(sequence number: 01, molecular weight: 5083, referring to Fig. 1)
Embodiment 2: the preparation of the segment (S-HP-2) with linker
The Start Fragment that embodiment 2 obtains reacts preparation with Fmoc-PEG4-NHS ester and has following structure segment S-HP-2.
The Start Fragment (S-HP) of 100nM is dissolved in the sodium borate buffer solution (pH=9.5,250mM) of 100 μ L, it will
The Fmoc-PEG4-NHS ester of 200nM is dissolved in 20 μ L dimethyl acetamides (DMAc) and the solution of S-HP is added at room temperature
In react at room temperature, after reaction, to reaction solution be added 12 μ L 5N sodium chloride solution and 360 μ L cold ethyl alcohol, -78 DEG C
4 DEG C of centrifugations again, remove supernatant after placing 0.5 hour, obtain DNA precipitating and continue to be dissolved in 2% aqueous piperidine solution, wait react
Quan Hou, ethanol precipitation as described above are purified to obtain S-HP-2 using HPLC.
The preparation of the DNA encoding library of compounds in the library embodiment 3:10 × 10
1, step (1): the connection of initial primers and segment S-HP-2 obtains the segment S-HP-2-P with primer
In this exemplary DNA encoding library of compounds, by a kind of nucleotide double of regular coding, (referred to as starting is drawn
Object, the customization of Suzhou Jin Weizhi Biotechnology Co., Ltd, HPLC purifying) it is connected to S-HP-2, it is compiled for subsequent all DNA of screening
The universal primer segment of the PCR of code compound:
Initial primers cochain: 5 '-PO4 2-- AGGCTAACTTGCGTACACAG-3 ' (sequence number: 02)
Chain under initial primers: 5 '-PO4 2-- ACGCAAGTTAGCCTTCGGGA-3 ' (sequence number: 03)
By the initial primers of the initial primers cochain of segment S-HP-2 aqueous solution 100 the μ L and 110nM of 100nM and 110nM
The 110 μ L of mixed aqueous solution of lower chain is uniformly mixed, then 95 DEG C of heating, 5 minutes (105 DEG C of the top cover temperature) in PCR instrument, then
It is cooled to room temperature with 1 DEG C of speed per second to anneal, N- cyanoimidazole, ZnCl is added later2And MgCl2, to fully reacting
Afterwards, be added 40 μ L 5N sodium chloride solution and 1200 μ L cold ethyl alcohol, -78 DEG C place 0.5 hour after again 4 DEG C centrifugation, removal
Supernatant obtains DNA precipitating and continues to be dissolved in 200 μ L distilled waters, the super filter tube (Amicon that the specification with 500 μ L of size is 10K
Ultra Centrifugal, article No.: Millipore-UFC501096) desalination and purification are carried out to product, it obtains with primer
Segment S-HP-2-P.
2. step (2): the synthesis of DNA encoding library of compounds first circulation
Similar to the Ligature of step (1), 10 kinds of connection reactions are provided with, are the segment that 1mM has primer by concentration
S-HP-2-P solution is dispensed into 10 continuous holes of 96 orifice plates, each 10 μ L of hole, continues that concentration is added to each hole to be 2mM
First circulation labeled nucleotide double-strand cochain and lower chain (referred to as first cycle labeling nucleotide double, Suzhou gold
The customization of Wei Zhi Biotechnology Co., Ltd, HPLC purifying) each 11nM, according to N- cyanoimidazole, ZnCl is added after step 1 annealing2
And MgCl2, after complete reaction, ethanol precipitation as described above obtains DNA precipitating and continues the sodium borate buffer solution for being dissolved in 10 μ L
In (pH=9.5,250mM), corresponding compound structural unity-small molecule amino acid is added and (it is new to be all from the bright Kant of Shanghai medicine
Rich net is look at by medicine development corporation, Ltd., 50 equivalents, the DMAc solution of 200mM) and 200mM 4- (4,6- dimethoxys equal three
Piperazine) -4- methyl morpholine hydrochloride (abbreviation DMT-MM, TCI, 45 equivalent, ready-to-use), after complete reaction, all reaction solutions
It is admixed together, ethanol precipitation is carried out as described above, is obtained DNA precipitating and is continued to be dissolved in 2% aqueous piperidine solution, to fully reacting
Afterwards, ethanol precipitation as described above obtains DNA precipitating and is dissolved in 200 μ L distilled waters, the ultrafiltration that the specification with 500 μ L of size is 10K
It manages (Amicon Ultra Centrifugal, article No.: Millipore-UFC501096) and desalination and purification is carried out to product, obtain
The product BB1-S-HP-2-P-T1 recycled to first.
First small molecule recycled and corresponding nucleotide double are specific as follows:
3, step (3): the synthesis of DNA encoding library of compounds second circulation
The labeled nucleotide double-strand (Suzhou Jin Weizhi Biotechnology Co., Ltd customization, HPLC purifying) of second circulation with
The link of the product BB1-S-HP-2-P-T1 of first circulation is similar to the Ligature (Fig. 2) of step 1, and DNA after purification is heavy
Shallow lake continues to be dissolved in the sodium borate buffer solution (pH=9.5,250mM) of 10 μ L, and small point of corresponding compound structural unity-is added
Sub- acid (being all from the rich net of looking at of Shanghai Yaoming Kangde New Medicine Development Co., Ltd, 50 equivalents, the DMAc solution of 200mM) and
4- (4,6- dimethoxy s-triazine) -4- methyl morpholine hydrochloride (abbreviation DMT-MM, TCI, 45 equivalent, now with existing of 200mM
With), after complete reaction, all reaction solutions are admixed together, carry out ethanol precipitation as described above, and it is molten to obtain DNA precipitating continuation
In 200 μ L distilled waters, super filter tube that the specification with 500 μ L of size is 10K (Amicon Ultra Centrifugal, article No.:
Millipore-UFC501096 desalination and purification) are carried out to product, obtain the product BB2-BB1-S-HP-2- of second circulation
P-T1-T2。
(sequence number: 44, average molecular weight: 31653, Fig. 3,4)
Second small molecule recycled and corresponding nucleotide double are specific as follows:
4, step (4): tail end primer is connect with DNA encoding library of compounds
In this exemplary DNA encoding library of compounds, by a kind of nucleotide double of regular coding, (abbreviation tail end draws
Object, the customization of Suzhou Jin Weizhi Biotechnology Co., Ltd, HPLC purifying) it is connected on DNA encoding library of compounds, as
The other end universal primer segment of PCR:
Tail end primer cochain: 5 '-PO4 2-- ATAGACTGCAAGCA-3 ' (sequence number: 45)
Chain under tail end primer: 5 '-PO4 2-- TGCAGATCTATTGTACG-3 ' (sequence number: 46)
The tail end primer cochain of DNA encoding library of compounds aqueous solution 10 μ L and 15nM of 10nM and the tail end of 15nM are drawn
The 15 μ L of mixed aqueous solution of chain is uniformly mixed under object, then 95 DEG C of heating, 5 minutes (105 DEG C of top cover temperature) in PCR instrument again, then
It is cooled to room temperature with 1 DEG C of speed per second to anneal, N- cyanoimidazole, ZnCl is added later2And MgCl2, to fully reacting
Afterwards, be added the sodium chloride solution of the 5N of 4 μ L and the cold ethyl alcohol of 120 μ L, -78 DEG C place 0.5 hour after 4 DEG C of centrifugations again, in removal
Clear liquid obtains DNA precipitating and continues to be dissolved in 50 μ L distilled waters, the super filter tube (Amicon that the specification with 500 μ L of size is 10K
Ultra Centrifugal, article No.: Millipore-UFC501096) desalination and purification, obtained DNA encoding are carried out to product
Library of compounds BB2-BB1-S-HP-2-P-T1-T2-CP can be used in the affine screening with multiple target point target.
Embodiment 4, DNA encoding library of compounds PCR efficiency compare
To DNA encoding library of compounds (BB2-BB1-S-HP-2-P-T1-T2-CP, the 5nM) solution for connecting tail end primer
TCEP (15 μ L, 100 equivalents) are added in (5 μ L), react 12 hours, and the sodium chloride solution of the 5N of 2 μ L and the cold second of 60 μ L is added
Alcohol, -78 DEG C place 0.5 hour after again 4 DEG C centrifugation, remove supernatant, obtain DNA precipitating continue to be dissolved in 50 μ L distilled waters, use
The specification of 500 μ L of size is super filter tube (Amicon Ultra Centrifugal, the article No.: Millipore- of 10K
UFC501096 desalination and purification) are carried out to product, and dilute obtained DNA fragmentation and the DNA encoding without cutting off using TCEP
The DNA of library of compounds to about 5 takes mole every microlitre, then does gradient series dilution respectively to two groups of DNA, passes through standard curve
Method imitates the amplification of PCR using Real-Time Fluorescent Quantitative PCR Technique (Quantitative Real-time PCR, abbreviation qPCR)
Rate is compared.Quantitative reagent is SYBR Green Master Mix (Cat#:Vazyme-Q141-02).
Experiments have shown that after cutting away the disulfide bond on Start Fragment with TECP, the PCR of the base sequence of left pairing
Efficiency with the base sequence for not using TCEP to act on comparatively, PCR efficiency improves 10%, the standard curve of amplification efficiency
See Fig. 5,6.
It is incorporated by reference into
The complete disclosure of herein cited each patent document and scientific literature is incorporated herein by reference for institute
Purposefully.
It is equivalent
The present invention can be implemented in other specific forms in the case where not departing from its essential characteristic.Therefore, aforementioned implementation
Example is considered illustrative, rather than the limitation to invention as described herein.The scope of the present invention is by appended claims
Book rather than indicated by aforementioned specification, and be intended to fall into all in the meaning and scope of the equivalents of the claims
Change is included therein.
Claims (40)
1. a kind of method for optimizing DNA encoding library of compounds Start Fragment, includes the following steps:
A) Start Fragment is provided, wherein the funtion part of the Start Fragment includes one or more reactive group R, and is risen
Another part of beginning segment connects oligonucleotide ssDNA;
B) funtion part of the Start Fragment and the structural unit containing at least one reaction site are covalently attached, wherein changing
The reaction site of object structural unit and the reactive group R of Start Fragment funtion part are closed to be covalently attached;
C) on the oligonucleotide ssDNA of the starting of the Start Fragment, the structural unit of markers step b) is capable of in introducing
Other coding oligonucleotides;
Wherein the structure I of the Start Fragment is as follows:
Wherein,
R is the reactive group of Start Fragment funtion part;
A is connection atom or skeleton structure;
S is the gasket for connecting R and A;
SsDNA is the oligonucleotide in step a);
B1、B2、B3、B4It is suitable for the functional group of A and oligonucleotide bonding;
L1, L2 are the attachments for connecting A and oligonucleotide ssDNA;
X is the site that can be cut off by mild condition shirtsleeve operation.
2. the method for claim 1, wherein the method also includes following steps:
D) DNA encoding library of compounds is combined with target albumen, collection can be with the library constructs of targeted integration;
E) coding oligonucleotide corresponding to the library constructs of step d) carries out PCR amplification and sequencing, determines energy and target
The structure for the library constructs that point albumen combines;
F) it is detached from DNA, and chemical synthesis is carried out to the library constructs that step e) is obtained, verifies its target validity.
3. method according to claim 1 or 2, wherein step b) and c) can sequentially exchange.
4. method according to claim 1 or 2, wherein step a) forms n times circulation to n times c) are repeated, and wherein n is 2
Above integer, preferably n are equal to 2 or 3 or 4.
5. method according to claim 1 or 2, wherein the reactive group R of Start Fragment funtion part is selected from ammonia in step a)
Base, hydroxyl, sulfydryl, carboxyl, nitrine, Terminal Acetylenes, alkynes, tetrazine, the trans- interior alkene of octatomic ring, aldehyde, ketone, halogenated aromatic ring or virtue in octatomic ring
Heterocycle, α-halogenatedketone or phosphorus ylide, the reactive group R of the Start Fragment funtion part are preferably amino or carboxyl.
6. method according to claim 1 or 2, wherein the reaction site of structural unit is anti-with Start Fragment funtion part
Answer group R to be covalently attached, the reaction site of the structural unit be selected from amino, carboxyl, sulfonyl, halogenated aromatic ring, heteroaromatic,
Isocyanate group, aldehyde, ketone or α, alpha, beta-unsaturated ketone or ester.
7. method according to claim 1 or 2, wherein the reaction site of structural unit is anti-with Start Fragment funtion part
Answer group R to be covalently attached, the reaction site of the structural unit is selected from phosphorus ylide, aldehyde or ketone.
8. method according to claim 1 or 2, wherein the reaction site of structural unit is anti-with Start Fragment funtion part
Answer group R to be covalently attached, the reaction site of the structural unit is selected from alkynes, tetrazine or eight yuan in nitrine and Terminal Acetylenes, octatomic ring
The trans- interior alkene of ring.
9. method according to claim 1 or 2, wherein the oligonucleotide ssDNA in step a) is single-stranded, and on
Chain and lower chain base sequence complementary match, while having pairing of the prominent base sequence for next code oligonucleotide to identify.
10. method according to claim 1 or 2, wherein the oligonucleotide of the starting in step c) and it is described other
Coding oligonucleotide is connected or is connected chemically by enzyme.
11. method according to claim 1 or 2, wherein the reactive group R of the Start Fragment funtion part not with starting
A, B and oligonucleotides ssDNA reaction in segment.
12. method according to claim 1 or 2, wherein the A is selected from-CH- or phloroglucin.
13. method according to claim 1 or 2, wherein the S is selected from oligoethylene glycol, lower alkylene, oligomeric N- first
Base-D-alanine, oligomeric N- methyl-L-alanine or oligomeric N- methyl DL-alanine.
14. such as the method for claim 13, wherein the S is 1~20 polyethylene glycol or 1~20 poly- N- methylalanine.
15. method according to claim 1 or 2, wherein the B1 be selected from phosphate, ester group, amido bond, carbamate,
Carbonate group or triazole, preferably phosphate;B2Selected from phosphate, ester group, amido bond, carbamate, carbonate group,
Or triazole, preferably phosphate;B3Selected from phosphate, ester group, amido bond, carbamate, carbonate group or triazole,
Preferably phosphate;B4Selected from phosphate, ester group, amido bond, carbamate, carbonate group or triazole, preferably phosphoric acid
Base.
16. method according to claim 1 or 2, wherein L1 is selected from oligoethylene glycol, lower alkylene, oligomeric N- methyl-
D-alanine, oligomeric N- methyl-L-alanine or oligomeric N- methyl DL-alanine;L2 is selected from oligoethylene glycol, lower
Base, oligomeric N- methyl-D-alanine, oligomeric N- methyl-L-alanine or oligomeric N- methyl DL-alanine.
17. the method described in claim 16, wherein L1 is 1~20 polyethylene glycol or 1~20 poly- N- methylalanine;L2
For 1~20 polyethylene glycol or 1~20 poly- N- methylalanine.
18. method according to claim 1 or 2, wherein X is that can be digested disconnected functional group, and the functional group is selected from two sulphur
Key.
19. such as the method for claim 18, wherein the enzyme for cutting off the disulfide bond is thiol reduction enzyme.
20. method according to claim 1 or 2, wherein X is can be by the functional group of chemical reaction cutting.
21. method according to claim 1 or 2, wherein X is the functional group that can be cut off by nucleopilic reagent or alkali, the function
Group structure be selected from by
Composed group.
22. method according to claim 1 or 2, wherein X is selected from the functional group of silicon atoms, the structure choosing of the functional group
Freely
Composed group.
23. such as the method for claim 22, wherein the reagent for cutting off the functional group of the silicon atoms is selected from fluorine anion, excellent
It is selected as tetrabutyl ammonium fluoride or hydrogen fluoride pyridine.
24. method according to claim 1 or 2, wherein X is the functional group that can be reduced reagent cutting, the functional group
Structure be selected from by
Composed group.
25. such as the method for claim 24, wherein the reagent for cutting off the hydrazine key is selected from samarium diodide, zinc/acetic acid, zinc/second
Alcohol, iron/acetic acid, palladium carbon/hydrogen, Raney's nickel/hydrogen or indium/ethyl alcohol.
26. such as the method for claim 24, wherein the reagent for cutting off the azo bond is selected from sodium dithionite, bisulfite
Sodium, palladium carbon/hydrogen, Raney's nickel/hydrogen, zinc/acetic acid or zinc/ethyl alcohol.
27. such as the method for claim 24, wherein the reagent for cutting off the nitrogen-oxygen bond is selected from samarium diodide, zinc/acetic acid, zinc/second
Alcohol, iron/acetic acid, palladium carbon/hydrogen, Raney's nickel/hydrogen or indium/ethyl alcohol.
28. such as the method for claim 24, wherein the reagent for cutting off the disulfide bond is selected from DTT, TCEP, triphenylphosphine, three fourths
Base phosphine, 2 mercapto ethanol, 1,3- dimercaptopropane, zinc/acetic acid or triphenylphosphine radium chloride/hydrogen.
29. such as the method for claim 24, wherein when the X is selected from disulfide bond, the oligonucleotides of the starting in step c)
Sour pass through with other described coding oligonucleotides is connected chemically.
30. method according to claim 1 or 2, wherein the described X functional group that can be cut off by organometallic reagent, it is described
The structure of functional group be selected from by
Composed group.
31. such as the method for claim 30, wherein the reagent for cutting off the allyl or benzyl is selected from four triphenyl phosphorus palladium/tri-
Ethylsilane or palladium carbon/hydrogen.
32. method according to claim 1 or 2, wherein the X is the functional group that can be oxidized cutting, the functional group
Structure be selected from by
Composed group.
33. such as the method for claim 32, wherein the reagent for cutting off the vicinal diamines is sodium metaperiodate.
34. such as the method for claim 32, wherein cutting off the β-seleno compound reagent is hydrogen peroxide or elemental iodine.
35. method according to claim 1 or 2, wherein the X is selected from the functional group that can be cut off by weak acid, the functional group
Structure be selected from by
Composed group.
36. such as the method for claim 35, wherein the reagent for cutting off the functional group is acetic acid or magnesium chloride.
37. method according to claim 1 or 2, wherein the X is selected from the functional group that can be cut off by light reaction, the function
Group structure be selected from by
Composed group.
38. method as claimed in claim 37, wherein the optical wavelength range for cutting off the functional group is selected from 254-532nm.
39. method according to claim 1 or 2, wherein L1, L2 can be simultaneously containing cut-off site X.
40. method according to claim 1 or 2, wherein X can L1 or L2 any one.
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