CN103882531B - A kind of synthesis of lead compound and screening method and test kit - Google Patents
A kind of synthesis of lead compound and screening method and test kit Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1068—Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
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- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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- C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
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- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/08—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
- C40B50/10—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support involving encoding steps
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- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B20/00—Methods specially adapted for identifying library members
- C40B20/04—Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
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Abstract
The invention discloses a kind of synthesis of lead compound and the method for screening, it comprises the steps:, and (1) gets raw material: get i kind building block and plant Single-stranded DNA fragments with (i+2); (2) with combinational chemistry, synthesis obtains the library of compounds of single stranded DNA mark, and wherein, initial building block fluorescence molecule marks; (3) screen: the library of the compound of DNA marker is screened; (4) check order: get the compound that step (3) screens the DNA marker obtained, to the DNA sequencing on the compound of DNA marker, building block and the reaction mechanism of this compound can be determined according to DNA sequence dna.The invention also discloses a kind of synthesis and screening reagent box of lead compound, and a kind of combinatorial chemistry library.The inventive method and test kit fast and effeciently can synthesize and screen and obtain object lead compound, monitor reaction process simultaneously, easy and simple to handle, with low cost.
Description
Technical field
The present invention relates to chemical field, particularly relate to a kind of synthesis of lead compound and screening method and test kit.
Background technology
From the latter stage eighties, along with the development of the breakthrough-High Throughput Screening Assay of molecular biology research, the growing number of the recruit's entity required for new drug development is many, scientists proceeds to from finding natural product compound group---the chemical libraries synthesizing high number attention, and chemical libraries is made up of the organic compound of many different attributes.Combinatorial chemistry synthesis method is a kind of technology of synthetic chemistry storehouse, uses this technology, and the building block of different series---reacted constituent lines up the diversified molecular entity group forming large series in an orderly manner.Combinatorial chemistry synthesis method is often called numbers game by people, namely how to arrange the problem of numerous building blocks.Theoretically, the total reaction product number N of combinatorial compound is determined by two factors, the building block number b of each step and the step x of synthesis, such as, linear combination for three steps is reacted, if the reactant number often walked is b1, b2, b3 respectively, the number of so theoretic total reaction product is N=b1b2b3.The target of combinatorial chemistry research is exactly all product N how effectively obtaining this reaction.In recent years, from solid phase synthesis to fast performance liquid parallel projects, combinatorial chemistry achieves breakthrough progress in synthetic method, conventional several synthetic methods have Solid-phase organic synthesis and liquid-phase organic synthesis, Solid-phase organic synthesis comprises mixing and splits point-score and parallel synthesis, and liquid-phase organic synthesis comprises multi component liquid phase synthesis method and functional group conversions's method.
In the chemical libraries that combinatorial chemistry synthetic technology is set up, product is thousands of, even more than one hundred million, and as classical organic synthesis, purifies and separates qualification is one by one no longer possible.High flux screening (Highthroughputscreening, HTS) technology refers to based on the experimental technique of molecular level and cell levels, using microplate format as experimental tool carrier, process of the test is performed with automation operating system, experimental result data is gathered with sensitive detecting instrument fast, carry out analyzing and processing with computer to experimental data, same time logarithm, in the sample detection of thousand, ten thousand, and supports with corresponding database the technical system that whole system operates.High-throughput screening method drastically increases speed and the efficiency of micromolecular compound screening, can screen the compound acting on target molecules from combinatorial chemistry library.But with traditional high-throughput screening method by after screening in compound chemically library, purifying also determines that the structure of target compound is very difficult, and the time of needs is long, and cost is high, and along with the expansion of library of compounds, difficulty is larger.
In order to solve this problem, application number: 95193518.6, denomination of invention: " the complex combination chemistry library with label coding " application discloses a kind of method, namely in each stage of synthesis, carry out on the carrier (such as particle) that synthesizes at compound, exclusively mark, to define in company with specific item (being often referred to added chemical reagent) adjoint during compou nd synthesis on this carrier.Described mark uses identifier molecule, and the sequenced item that between this point of subrecord synthesis phase, carrier granule experiences, provides the reaction mechanism of production compound on this carrier thus.But this application does not provide the technical scheme realizing the method.
Have report to use the synthesis unit of oligonucleotide marker compound in prior art, according to the general knowledge of biological field, under normal conditions, double-stranded DNA comparatively single stranded DNA is stablized, and therefore usually selects the synthesis unit of the oligonucleotide of double-strand to compound to mark, as:
Notification number: EP0643778, denomination of invention: the method that patent discloses with single stranded oligonucleotide labeled amino acid or polypeptide of " encodedcombinatorialchemicallibraries "; Notification number: US7935658, denomination of invention: " methodsforsynthsisofencodedlibraries " patent discloses the building block of one double chain DNA fragment labelled synthesis, forms the method for library of compounds; Application number: WO/2010/094036, denomination of invention: oligonucleotide marker compound is used in application discloses of " METHODSOFCREATINGANDSCREENINGDNA-ENCODEDLIBRARIES ", form the method for library of compounds, its oligonucleotide is the double-stranded DNA of hairpin structure.
But, during with the building block of double-stranded DNA labelled synthesis or compound, in connection extension process, double-stranded DNA is easily cross-linked, form curling tertiary structure, during order-checking, needs unwind, operate more complicated, when marking the linear combination reaction of more than three steps with double-stranded DNA, the sequencing result error of double-stranded DNA is larger, the method is caused to rest on bidimensional, thus can only increase compound amounts in library by the building block number increasing every single step reaction, obtained library of compounds diversity is poor, and not easily synthesis obtains target compound.
Need searching new, easy and simple to handle, result is marking method more accurately.
Summary of the invention
In order to solve the problem, the invention provides a kind of synthesis of lead compound and the test kit of screening and method, and a kind of new combinatorial chemistry library.
Explanation of nouns:
Building block (SyntheticBuildingBlock), is synthon again, refers to micromolecular compound that possess various physico-chemical property and particular organisms chemical property, that must use in new drug (Western medicine, agricultural chemicals) R&D process.
Lead compound (leadcompound) is called for short primer, is the compound with certain biological activity and chemical structure obtained with means by all means, for further structure of modification and modification, is the starting point of modern new drug research.
Reaction mechanism: react the process experienced exactly.
Be connected in series: refer to that between some sections of single-stranded DNA sequence, two-end-point is connected successively, and tie point does not have branch.
The synthesis of lead compound of the present invention and the method for screening, it comprises the steps:
(1) get raw material: i kind building block and (i+2) plant Single-stranded DNA fragments, and (i+2) plants Single-stranded DNA fragments and comprise i kind flag sequence, a kind of top sequence and a kind of end sequence, i kind flag sequence is specific mark i kind building block respectively, wherein, i=1,2,3 ... n;
(2) combinational chemistry synthetic compound is used:
A, prepare initial building block: select 1 ~ i kind building block fluorescent mark, again one end of top sequence is connected on building block, the specific tagging sequences of the other end and described building block is connected in series, and obtains the initial building block that 1 ~ i kind is marked with the single stranded DNA of an end dissociative;
Based on b, the initial building block that obtains by step a, with the mode synthetic compound of linear combination reaction, in building-up process, often add new building block, just be connected in series the specific tagging sequences of added building block at the single stranded DNA free end be connected with initial building block, described single stranded DNA is extended, after end of synthesis gradually, be connected in series end sequence at described single stranded DNA free end, namely obtain the library of compounds of single stranded DNA mark;
(3) screen: the library of the compound of DNA marker is screened, selects target compound;
(4) check order: DNA step (3) being screened the target compound obtained checks order, and determines the structure of target compound.
Wherein, step (1) described top sequence comprises polyadenosine.Preferably, described polyadenosine is 12 ~ 20 adenosines.
Wherein, the length of step (1) described flag sequence is not less than 6bp.Preferably, the length of described flag sequence is 9 bp.
Wherein, in described step (2) building-up process, pH is 8 ~ 12, and temperature is 0 ~ 30 DEG C.
Wherein, 3 ' end connection ribonucleotide of step (1) described flag sequence.Described ribonucleotide is cytidine.
In step (2), described in a step, fluorescent mark is: get initial building block and fluorescence molecule, connects.
Described fluorescence molecule is 7-aminocoumarin-3-carbonic acid Acibenzolar, 7-diethylin tonka bean camphor-3-carbonic acid Acibenzolar, 7-aminocoumarin-3-carbonic acid Acibenzolar, ayapanin-3-carbonic acid Acibenzolar, 5/6-fluorescein carbonic acid Acibenzolar, fluorescein-5-lsothiocyanates (FITC), fluorescein-5 maleimide, 5-iodacetyl amido fluorescein, rhodamine 6G-5/6-carbonic acid Acibenzolar, Sulforhodamine B SULPHURYL CHLORIDE, X-rhodamine-6-carbonic acid Acibenzolar, Sulforhodamine 101 SULPHURYL CHLORIDE, four ethylamino rhodamine-5/6-carbonic acid Acibenzolars, four methylamino-rhodamine-6-maleimides, Dallas Pink C2 maleimide, four methylamino-rhodamine-5/6-lsothiocyanates, four methylamino-rhodamine-5/6-maleimides, dansyl chloride, EDANSC2 maleimide, iodacetyl amido EDANS, NBD-X Acibenzolar, ThioGlo-1 or CHROMISL5HNazide.
Wherein, in step (2), the method that in a step, top sequence is connected with initial building block is:
By top sequence amination, initial building block carboxyl, sulfydryl or alkynyl, react and get final product.
Wherein, between step (2) described connection top sequence and flag sequence, flag sequence or the method for attachment of flag sequence and end sequence be: make 5 ' of single stranded DNA with many nucleoside kinases-hold phosphorylation, connect by RNA ligase.Described many nucleoside kinases are the many nucleoside kinases of T4, and described RNA ligase is T4RNA ligase enzyme.
Wherein, the screening method in described step (3) is the screening method based on receptor-ligand specific reaction.
The synthesis of lead compound of the present invention and screening reagent box, it comprises following composition:
1) i kind building block and (i+2) plant Single-stranded DNA fragments, and Single-stranded DNA fragments is divided into top sequence, end sequence and i kind flag sequence, and i kind flag sequence is specific mark i kind building block respectively, wherein, and i=1,2,3 ... n;
2) fluorescent mark reagent, top sequence are connected with building block and connect with reagent with reagent, combinational chemistry reagent and Single-stranded DNA fragments;
3) screening compound reagent;
4) DNA sequencing reagent.
Wherein, composition 1) described top sequence comprises polyadenosine.Preferably, described polyadenosine is 12 ~ 20 adenosines.
Wherein, composition 1) length of described flag sequence is not less than 6bp.Preferably, the length of described flag sequence is 9 bp.
Wherein, 3 ' end connection ribonucleotide of step (1) described flag sequence.Described ribonucleotide is cytidine.
Wherein, composition 2) described fluorescent mark reagent comprises fluorescence molecule.
Described fluorescence molecule is 7-aminocoumarin-3-carbonic acid Acibenzolar, 7-diethylin tonka bean camphor-3-carbonic acid Acibenzolar, 7-aminocoumarin-3-carbonic acid Acibenzolar, ayapanin-3-carbonic acid Acibenzolar, 5/6-fluorescein carbonic acid Acibenzolar, fluorescein-5-lsothiocyanates (FITC), fluorescein-5 maleimide, 5-iodacetyl amido fluorescein, rhodamine 6G-5/6-carbonic acid Acibenzolar, Sulforhodamine B SULPHURYL CHLORIDE, X-rhodamine-6-carbonic acid Acibenzolar, Sulforhodamine 101 SULPHURYL CHLORIDE, four ethylamino rhodamine-5/6-carbonic acid Acibenzolars, four methylamino-rhodamine-6-maleimides, Dallas Pink C2 maleimide, four methylamino-rhodamine-5/6-lsothiocyanates, four methylamino-rhodamine-5/6-maleimides, dansyl chloride, EDANSC2 maleimide, iodacetyl amido EDANS, NBD-X Acibenzolar, ThioGlo-1 or CHROMISL5HNazide.
Wherein, composition 2) described top sequence is connected with building block by pack containing the reagent of amination single stranded DNA and the reagent of the initial building block of carboxyl, sulfydryl or alkynyl.
Wherein, composition 2) described Single-stranded DNA fragments connection reagent comprises multinuclear glycosides kinases and RNA ligase.
Preferably, described many nucleoside kinases are the many nucleoside kinases of T4, and described RNA ligase is that T4RNA ligase enzyme connects.
Combinatorial chemistry library of the present invention, it take building block as raw material, with the combinatorial chemistry library of combinational chemistry synthesis, wherein, each compound label one section of single-stranded DNA sequence and a fluorescence molecule, the structure of this single-stranded DNA sequence is top sequence---i kind flag sequence---end sequence, the building block used in described i kind flag sequence specific mark i kind combinatorial chemistry building-up process, it puts in order identical with the addition sequence of building block in combinatorial chemistry building-up process.
Wherein, the length of described flag sequence is not less than 6bp.Preferably, the length of described flag sequence is 9 bp.
When the length of flag sequence is 6,4096 not homotactic Single-stranded DNA fragments can be prepared, DNA fragmentation coding, thousands of for the preparation of the building block in combinatorial chemistry library, the needs of most compounds synthesis and screening can be met; Flag sequence be or 9 time, 262144 not homotactic Single-stranded DNA fragments can be prepared, its coding, reach 262144 for the preparation of the building block in combinatorial chemistry library, DNA fragmentation coding, millions of for the preparation of the building block in combinatorial chemistry library, the needs of compou nd synthesis and screening can be met completely.If flag sequence is longer, its building block quantity that can encode is larger, and the combinatorial chemistry library of preparation is larger, but accordingly, cost is higher, considers storage capacity and cost, optimum when the length of flag sequence is 9.
Under condition of the present invention, with the building block of single stranded DNA labelled synthesis, in connection procedure, can not be complementary and form double-strand, Stability Analysis of Structures between single stranded DNA, be also not easily cross-linked, do not need during order-checking to unwind, simple to operate, quick, result is accurate.Therefore, the inventive method can comprise multi-step linear composite reaction, and the library of compounds diversity of synthesis is good, and storage capacity is large, easy synthesis obtains target compound and determines its building block, reaction mechanism and chemical structure, thus synthesis obtains a large amount of target compound rapidly.
Initial building block marks fluorescence molecule, fluorescent imaging is carried out after being connected with top sequence, using top sequence silver dye band as standard, by determining whether the two have band in same position, the whether successful connection of initial building block and top sequence namely accurately can be judged; Similarly, in subsequent synthetic procedures, fluorescent imaging is carried out to the compound of labeled ssdna molecule, using identical single strand dna silver dye band as standard, by determining whether the two has band in same position, accurately can judge whether compound is connected with single strand dna.Therefore, whether can marked DNA molecular by fluorescent mark deterministic compound and intermediate thereof, monitoring reaction process, avoids the wasting of resources.
The inventive method is a kind of precise and high efficiency, lead compound library easy and simple to handle, with low cost synthesis and screening method, and application prospect is good.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following embodiment.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
Accompanying drawing explanation
Fig. 1 the present invention process schematic diagram of combinational chemistry synthetic compound, wherein, " H " represents building block; " just " represents initiation sequence; " B " expressive notation sequence, its specific mark building block, the corresponding relation of both digitized representations, e.g., B1 specific mark H1; " end " represents end sequence; Left hurdle represents reactions steps, consistent with embodiment 1 reactions steps; The end product obtained, building block only represents the addition sequence of building block from right to left, and initiation sequence, flag sequence and end sequence represent the structure of the single-stranded DNA sequence finally obtained from left to right;
Fig. 2 chemistry library fluorescence imaging of the present invention result.Mapping is conventional silver dye result, and right figure is fluorescence imaging result, swimming lane 1:DNAMarker; Swimming lane 2: with the reaction of EDCI as activator.
The electrophorogram of the trypsin inhibitor that Fig. 3 chemistry library of the present invention and screening obtain;
Fig. 4 sequencing result histogram, post and compound one_to_one corresponding, its height is relevant to the bonding force of this compound and target;
The IC50 collection of illustrative plates of Fig. 5 trypsin inhibitor of the present invention;
The IC50 collection of illustrative plates of Fig. 6 trypsin inhibitor of the present invention.
Embodiment
Form by the following examples sets forth embodiment, and is described in further detail foregoing of the present invention.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following embodiment.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
The synthesis of embodiment 1 lead compound of the present invention and screening method
1, preparation method
(1) building block and Single-stranded DNA fragments is got:
I kind building block and (i+2) plant Single-stranded DNA fragments, and (i+2) plants Single-stranded DNA fragments and comprise i kind flag sequence, a kind of top sequence and a kind of end sequence, and i kind flag sequence is specific mark i kind building block respectively, wherein, and i=1,2,3 ... n;
Following flag sequence all can connect cytidine, improve the joint efficiency of follow-up Single-stranded DNA fragments.
Table 1 Single-stranded DNA fragments
(2) synthesize: as shown in Figure 1, with combinational chemistry synthetic compound library:
a, prepare initial building block: select 1 ~ i kind building block fluorescent mark, by top sequence
one end is connected on building block, and the specific tagging sequences of the other end and described building block is connected in series,
obtain the initial building block that 1 ~ i kind is marked with the single stranded DNA of an end dissociative, e.g., i=2;
1. initial building block is connected with top sequence:
Get top sequence, amination;
Get building block 1 and 2, (fluorescence molecule is 7-aminocoumarin-3-carbonic acid Acibenzolar to use fluorescent mark respectively, 7-diethylin tonka bean camphor-3-carbonic acid Acibenzolar, 7-aminocoumarin-3-carbonic acid Acibenzolar, ayapanin-3-carbonic acid Acibenzolar, 5/6-fluorescein carbonic acid Acibenzolar, fluorescein-5-lsothiocyanates (FITC), fluorescein-5 maleimide, 5-iodacetyl amido fluorescein, rhodamine 6G-5/6-carbonic acid Acibenzolar, Sulforhodamine B SULPHURYL CHLORIDE, X-rhodamine-6-carbonic acid Acibenzolar, Sulforhodamine 101 SULPHURYL CHLORIDE, four ethylamino rhodamine-5/6-carbonic acid Acibenzolars, four methylamino-rhodamine-6-maleimides, Dallas Pink C2 maleimide, four methylamino-rhodamine-5/6-lsothiocyanates, four methylamino-rhodamine-5/6-maleimides, dansyl chloride, EDANSC2 maleimide, iodacetyl amido EDANS, NBD-X Acibenzolar, ThioGlo-1 or CHROMISL5HNazide), carboxyl again, sulfydryl or alkynyl,
Carboxyl, sulfydryl or alkynyl; Get the building block 1 and 2 of activation and the top serial response of activation, the initial building block of top sequence must be connected to;
2. in top sequence, connect the flag sequence (this method of attachment except can with except following method, can also with the method for attachment of other single stranded DNAs) of building block 1 and 2 respectively:
5 '-end phosphorylation of single stranded DNA is made with many nucleoside kinases; RNA ligase connects;
3. mix, obtain initial building block mixture.
based on b, the initial building block that obtains by step a, by the mode synthesis of linear combination reaction
compound, in building-up process, often adds new building block, just at the strand be connected with initial building block
dNA free end is connected in series the specific tagging sequences of added building block, make described single stranded DNA by
gradually extend, after end of synthesis, be connected in series end sequence at described single stranded DNA free end, namely obtain
the library of compounds of single stranded DNA mark; Such as, three step linear combination reactions.
I：
1. synthesis is (except following synthetic method, other chemical synthesis process can also be used): get building block 3 ~ 4, be placed in 2 micro-scale reaction vessels, mix in initial building block mixture obtained with step a respectively, split the synthesis of point-score, parallel synthesis, multi component liquid phase synthesis method or functional group conversions's method according to mixing;
2. to label sequence: with step a step 2..
3. mix, obtain mixture.
Ⅱ：
1. synthesis is (except following synthetic method, other chemical synthesis process can also be used): get building block 5 ~ 6, be placed in 2 micro-scale reaction vessels, obtain mixture with step b respectively to mix, split the synthesis of point-score, parallel synthesis, multi component liquid phase synthesis method or functional group conversions's method according to mixing;
2. to label sequence: with step a step 2..
3. end sequence is added: with step a step 2..
4. mix, namely
obtain the library of the compound of single stranded DNA mark.
(3) screen: the library of the compound of DNA marker is screened:
Based on the chromatographic separation screening method of receptor-ligand specific reaction, screen with the library of bio-target molecule to the compound of DNA marker.
Wash-out is carried out to chromatogram, the compound of the DNA marker be not combined with biological target molecule is removed, be separated the compound obtaining the DNA marker be combined with bio-target molecule.
(4) check order:
Get the compound that step (3) screens the DNA marker obtained, to the DNA sequencing on the compound of DNA marker, building block and the reaction mechanism of this compound can be determined according to DNA sequence dna.
Embodiment 2 becomes by the inventive method and screens the lead compound of trypsin inhibitor
1, material and reagent
T4PNK(500UNEB-M0201V),T4RNAligase1(NEB-M0204S),Cartridges(PCRpurificationKit(cat.no28104,NucleotidesremovalKitcat.no28306)purchasedfromQiagen(Hilden,Germany).dNTPs(0.5mM,NEB,cat.no89009).
Single-stranded DNA fragments shown in table 1, is synthesized by Genscript company and Biosune company.
2, preparation method
(1) Single-stranded DNA fragments is prepared:
55 kinds of building blocks and 57 kinds of Single-stranded DNA fragments, 57 kinds of Single-stranded DNA fragments comprise 55 kinds of flags sequence, a kind of top sequence and a kind of end sequence;
Following flag sequence all can connect cytidine, improve the joint efficiency of follow-up Single-stranded DNA fragments.
Table 2 Single-stranded DNA fragments
(2) synthesize:
a, prepare initial building block: select a kind of building block fluorescent mark, by one of top sequence
end is connected on building block, and the specific tagging sequences of the other end and described building block is connected in series,
to a kind of initial building block being marked with the single stranded DNA of an end dissociative;
1. initial building block is connected with top sequence:
Get top sequence, amination, get building block 1 FITC and mark, carboxylated; Get the synthesis of activation
building block 1 and the top serial response of activation, must be connected to the initial building block of top sequence.
Fluorescent mark: by building block 1(3.0mg, 0.023mmol) be dissolved in 1.0mL dimethyl sulfoxide (DMSO), be added dropwise to fluorescein isothiocyanate FITC(7.5mg, 0.019mmol under room temperature) 1.0mL dimethyl sulphoxide solution.Reaction mixture is at room temperature after reaction overnight, by high performance liquid chromatography separation and purification.
Connect: reaction mixture cumulative volume is 150 microlitres, solvent is water and methyl-sulphoxide volume ratio is 3:7, wherein containing triethylamine salt acid buffering system (pH10.0,80mM), wherein, the concentration of building block 1 is 30mM, activating reagent 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride (EDCI) concentration is 4mM, and 2-sulfonic group-N-hydroxy-succinamide concentration is 10mM, top sequence concentration is 20M, room temperature reaction 1 hour.
2. in top sequence, connect the flag sequence (this method of attachment except can with except following method, can also with the method for attachment of other single stranded DNAs) of building block 1:
5 '-end phosphorylation of single stranded DNA is made with many nucleoside kinases; RNA ligase connects.
Connect: get step 1. process after top sequence and the reaction mixture of flag sequence 1,15ul comprise the damping fluid of 225pmol top sequence, 25pmol flag sequence 1,50 unit T4RNA ligase enzyme and ligation; This mixture hatches 1.5h at 25 DEG C, after 70 DEG C of heating 20min, T4RNA ligase enzymes sex change again; Add T4 polynucleotide kinase and 1nmATP, react 10 circulations, then hatch 20min at 75 DEG C and make extra many nucleoside kinases sex change;
Purifying: product is with in isopyknic 2 × sample-loading buffer, and this damping fluid comprises 40mMTris-HCL (pH7.6), 1MNaCL and 1mMEDTA;
The mixture obtained purifying as follows: QiagenCartridge pillar on reaction solution; Suspend with 1 × sample-loading buffer; The centrifugal 1min of 1000rmp, filters with siliceous glass cotton; Rinse successively with 1 × sample-loading buffer, 0.5MNaCL solution and 80% ethanol; Use 20ulPE elution; Vacuum-drying.
Detect: detect with conventional silver dye electrophoresis and fluorescence imaging, fluoroscopic image and silver dye image are alignd, the image of silver dye finds corresponding band by the molecular weight contrast of DNA before DNAMarker and reaction.If band appears in the corresponding position on fluoroscopic image, the covalently bound success of small molecules-DNA is described, the power of fluorescence response shows to react the quality of carrying out.As shown in Figure 2, there is band in the corresponding position on fluoroscopic image in detected result, the covalently bound success of small molecules-DNA.Experimental result illustrates, can with the initial building block of fluorescent mark, and then whether monitoring compound and intermediate thereof marked DNA molecular, avoid the wasting of resources.
based on b, the initial building block that obtains by step a, close by the mode of three step linear combinations reactions
become compound, in building-up process, often add new building block, to be just connected with initial building block
single stranded DNA free end is connected in series the specific tagging sequences of added building block, makes described strand
dNA extends gradually, after end of synthesis, is connected in series end sequence at described single stranded DNA free end,
namely the library of compounds of single stranded DNA mark is obtained;
first building block, i.e. initial building block (1): building block 1;
second batch building block (5): building block 2 ~ 6;
3rd batch of building block (49): building block 7 ~ 55;
I：
1. synthesize
Get building block 2 ~ 6, be placed in 5 micro-scale reaction vessels respectively, the initial building block obtained with step a mixes, and splits the synthesis of point-score, parallel synthesis, multi component liquid phase synthesis method or functional group conversions's method according to mixing.
Be placed in 5 micro-scale reaction vessels respectively, the initial building block obtained with step a reacts.For building block 2, reaction conditions is 150 microlitre reaction mixtures, solvent is water and methyl-sulphoxide volume ratio is 3:7, wherein containing triethylamine salt acid buffering system (pH9.0,80mM), the concentration of building block 1 is 30mM, activating reagent 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride (EDCI) 4mM, 2-sulfonic group-N-hydroxy-succinamide 10mM, the concentration 1.5M of building block 2, room temperature reaction 15 hours.
2. the flag sequence of building block 2 ~ 6 is added respectively: with step a step 2..
3. mix, obtain mixture.
Ⅱ：
1. synthesize
Get building block 7 ~ 55, be placed in 49 micro-scale reaction vessels respectively, the initial building block obtained with step a mixes, and splits the synthesis of point-score, parallel synthesis, multi component liquid phase synthesis method or functional group conversions's method according to mixing.
Be placed in 49 micro-scale reaction vessels respectively, the initial building block obtained with step a reacts.For building block 2, reaction conditions is 150 microlitre reaction mixtures, solvent is water and methyl-sulphoxide volume ratio is 3:7, wherein containing triethylamine salt acid buffering system (pH9.0,80mM), the concentration of building block 1 is 30mM, activating reagent 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride (EDCI) 4mM, 2-sulfonic group-N-hydroxy-succinamide 10mM, the concentration 1.5M of building block 2, room temperature reaction 15 hours.
2. to label sequence: with step a step 2..
3. end sequence is added: with step a step 2..
4. mix, namely
obtain the library of the compound of single stranded DNA mark.
(3) screen: the library of the compound of DNA marker is screened:
Based on the chromatographic separation screening method of receptor-ligand specific reaction, screen with the library of bio-target molecule to the compound of DNA marker.
1. CNBr is resin activated:
1) 0.1033 gram of CBNr activates Sepharose4B resin, and they are divided into 2, then inner static at the hydrogen chloride solution (PH3.0) of the 1mM of 4ml;
2) 15 minutes are cleaned with hydrochloric acid (pH value 3.0) scavenging solution of 1mM;
3) trypsinase of 4mg is dissolved in the coupling buffer (0.1M sodium bicarbonate, 0.5M sodium-chlor, pH value 8.3) of 0.5 milliliter;
4) the slight mixture of concussion up and down 1 hour, in room temperature or 4 DEG C of night incubation;
5) unnecessary protein is washed away with 4ML coupling solution;
6) resin is proceeded in the Tris-HCl damping fluid (pH8.0) of 4mL0.1M, hatch 2 hours;
7) resin is cleaned three times with cleaning buffer solution 1 and 2; (scavenging solution 1:0.1Maceticacid, 0.5MNaCl, pH4.0; , scavenging solution 2:0.1MTris-HCl, 0.5MNaCl, pH8.0.)
8) centrifugation resin, 6000r/min, 10min.
2. the solidification of trypsinase on activation CNBr resin
1) take in the hydrochloric acid of 100 milligrams of CNBr resins activated as 4 milliliters of 1mM and hatch;
2) clean with the 1mM hydrochloric acid (pH value 3.0) of 8mL;
3) trypsin solution of 0.004mg/ml, 0.02mg/ml, 0.1mg/ml, 0.5mg/ml, 2.5mg/ml is activated CNBr mixed with resin with five parts respectively, at 4 DEG C, hatch 5 hours;
4) 0.1MTris hydrochloric acid is used, 0.5M sodium-chlor, (pH value 8.3) solution cleaning resin;
5) 0.1M sodium-acetate is used, 0.5M sodium-chlor, (pH4.0) solution cleaning resin;
6) 4,5 steps are repeated, alternately cleaning at least 3 circulations.
7) resin that trypsinase solidifies is kept at (pH7.4) in PBS damping fluid at 4 DEG C;
3. trypsinase compound storehouse affinity selection
1) library of the compound of the single stranded DNA mark that the step (2) of getting obtains mixes (17uL:255uL) with 1:15 volume ratio with PBS damping fluid;
2) respectively the library sample of 50 μ L is added pancreas bovine trypsin/CNBr paste resin (2.5,0.5,0.1,0.02,0.004 and 0mg/mL);
3) with the herring sperm dna solution of PBS buffer 0.3 mg/ml;
4) the herring sperm dna solution that obtains of step 3) and step 2) pancreas bovine trypsin/CNBr paste resin of obtaining hatches 1 hour at 25 DEG C;
5) mixture of step 4) is transferred in the Spin pillar of 2ml, removing supernatant liquor;
6) with 200 μ LPBS buffer solution resins, repeat 4 times;
7) add 100 μ L sterilized waters in slurries after cleaning, screening obtains and trypsinase ligand sample.
Qualification: get the library of compounds of the single stranded DNA mark that step (2) obtains and step (3) and screen the affine sample electrophoresis of the trypsinase obtained and detect.
As shown in Figure 3, all obtain a target stripe with different concns pancreas bovine trypsin/CNBr paste resin screening, negative control is then blank band to detected result, illustrates that the present invention's screening obtains the trypsinase ligand sample of purifying.
(4) check order:
Get the compound that step (3) screens the DNA marker obtained, to the DNA sequencing on the compound of DNA marker, building block and the reaction mechanism of this compound can be determined according to DNA sequence dna:
Get step (3) to screen the sample obtained and carry out polysaccharase Ligature (PCR), the oligonucleotide code of coded compounds is carried out pcr amplification (cumulative volume 50 microlitre, 30 circulations, each circulation 94 DEG C 1 minute, 55 DEG C are reacted 1 minute, 72 DEG C are reacted 40 seconds), with 5 μ L trypsinase 245 storehouse (concentration 100fM) for template.
Adopt IlluminaHiseq2000 high-flux sequence platform, order-checking flow process is as follows:
1) the screening oligonucleotide library after pcr amplification, utilizes the MAG-PCR-CL-250 test kit of Axygen company to carry out purifying and quality examination report;
2) utilize the Picogreen test kit of Illumina company to carry out nucleic acid quantification, draw sample nucleic concentration, carry out next step sequencing library preparation;
3) the chip-seqDNAsample test kit of Illumina company is utilized specific for Hiseq2000 sequence measuring joints (6 bases longs) to be connected on 5 ' end and 3 ' ends of order-checking sample, above the chip chip-seqplate being fixed to Hiseq2000 sequenator again, carry out next step bridge amplification;
4) utilize TruseqPEClusterKitv3-cBot-HS test kit to carry out sample of nucleic acid bridge amplification, on each chip-seqlane, obtain the nucleic acid bunch (cluster) that enough order-checkings use;
5) utilize the laserimaging system of Hiseq2000, the tape label dNTP of TruseqSBSKitv3-HS (200cycles), record each base appearance order and the frequency that read from sequence measuring joints, test sample of nucleic acid base;
6) under, machine takes out data, data processing.
As shown in Figure 4, sequence is as shown in SEQIDNO.1 for sequencing result:
TCAGGCAGAGGCGATAGAGGCGATAGA, associative list 2 can determine that the structure of screening the trypsinase part obtained is as follows:
According to this structural formula, detect after synthesizing this compound, detect and determine that this compound is trypsin inhibitor, its Inhibiting enzyme activity is as shown in Fig. 5 ~ 6, and IC50 is 8.1 ± 2.1nM, illustrates that screening the compound obtained is trypsinase part really.
Experimental result explanation, the present invention constructs a chemistry library containing 245 compounds, and screening obtains a trypsinase part, it has the tryptic activity of suppression, illustrate that the inventive method can effectively be synthesized and screen lead compound, fluorescent mark can effectively monitor whole building-up process.
The synthesis of embodiment 3 lead compound of the present invention and screening reagent box
1, the composition (the synthesis consumption of N number of building block) of test kit of the present invention
1) i kind building block and i+2 kind Single-stranded DNA fragments, is divided into top sequence, end sequence and i kind flag sequence by Single-stranded DNA fragments, i kind flag sequence is the code of i kind building block respectively, wherein, and i=1,2,3 ... n;
Table 3 building block and not homotactic Single-stranded DNA fragments
2) top sequence is connected with building block to synthesize with reagent, combinational chemistry and connects with reagent with reagent and Single-stranded DNA fragments;
Table 4 top sequence is connected with building block uses reagent
| Reagent name | Consumption |
| Triethylamine salt acid buffer, pH10.0 | （800mM，15ul） |
| 1-ethyl-3-(3-dimethylamine propyl) carbodiimide | （40mM，15ul） |
| 2-sulfonic group-N-hydroxy-succinamide | （100mM，15ul） |
Table 5 fluorescent mark reagent
| Reagent name | Consumption |
| Dimethyl sulfoxide (DMSO) | Volume: 2ml |
| Fluorescein isothiocyanate FITC | （7.5mg,0.019mmol） |
Table 6 combinatorial chemistry synthetic method reagent
| Reagent name | Consumption |
| Triethylamine salt acid buffer, pH9.0 | （800mM，15ul） |
| 1-ethyl-3-(3-dimethylamine propyl) carbodiimide | （40mM，15ul） |
| 2-sulfonic group-N-hydroxy-succinamide | （100mM，15ul） |
Table 7DNA fragment connection reagent
| Reagent name | Consumption |
| T4PNK(10U/ul) | N×10ul |
| 10×T4 RNA ligase buffer | N×10ul |
| ddH2O | N×77.4ul |
| T4RNA ligase(10U/ul) | N×10ul |
| 10×T4 RNA ligase buffer | N×2.5ul |
| ATP(10mM) | N×0.1ul |
3) screening compound reagent;
Table 8 screening compound reagent
4) DNA sequencing reagent.
Table 9DNA order-checking reagent
| Purposes | Reagent name |
| PCR purifying | MAG-PCR-CL-250 |
| Nucleic acid quantification | Picogreen test kit |
| Library construction | chip-seq DNA sample kit |
| Bridge amplification | Truseq PE Cluster Kit v3-cBot-HS |
| Upper machine order-checking | Truseq SBS Kit v3-HS(200cycles) |
The method that test kit of the present invention provides according to the embodiment of the present invention 1 uses, and can be used for Fast back-projection algorithm and the screening of lead compound.
To sum up, with prior art with compared with the building block of double-stranded DNA labelled synthesis, the present invention's single stranded DNA labelled synthesis building block, in connection procedure, single stranded DNA can not be complementary, not easily be cross-linked, Stability Analysis of Structures, pcr amplification and the order-checking of single stranded DNA are more convenient compared with double-stranded DNA, quick; After screening obtains target compound, the single-stranded DNA sequence of target-marking compound can be determined easily and fast, accurately, thus determine the building block of its target compound marked, reaction mechanism and chemical structure, greatly improve screening efficiency; Initial building block marks fluorescence molecule, and whether deterministic compound and intermediate thereof marked DNA molecular, and monitoring reaction process, avoids the wasting of resources.
The synthesis of lead compound of the present invention and screening method and test kit precise and high efficiency, easy and simple to handle, with low cost, have a good application prospect.
Claims (21)
1. the synthesis of lead compound and a screening method, is characterized in that: it comprises the steps:
(1) raw material is got: i kind building block and (i+2) plant Single-stranded DNA fragments, (i+2) plant Single-stranded DNA fragments and comprise i kind flag sequence, a kind of top sequence and a kind of end sequence, i kind flag sequence is specific mark i kind building block respectively, wherein, i=1,2,3 ... n;
(2) with combinational chemistry synthetic compound library:
A, prepare initial building block: select 1 ~ i kind building block fluorescent mark, again one end of top sequence is connected on building block, the specific tagging sequences of the other end and described building block is connected in series, and obtains the initial building block that 1 ~ i kind is marked with the single stranded DNA of an end dissociative;
B, the initial building block obtained with step a, with 1 ~ i kind building block, with the mode synthetic compound of linear combination reaction, in building-up process, often add new building block, be just connected in series the specific tagging sequences of added building block at the single stranded DNA free end be connected with initial building block, described single stranded DNA is extended gradually, after end of synthesis, be connected in series end sequence at described single stranded DNA free end, namely obtain the library of compounds of single stranded DNA mark;
(3) screen: the library of the compound of DNA marker is screened, selects target compound;
(4) check order: DNA step (3) being screened the target compound obtained checks order, and determines building block and the reaction mechanism of target compound;
3 ' end connection ribonucleotide of step (1) described flag sequence; Described ribonucleotide is cytidine;
Step (1) described top sequence comprises polyadenosine.
2. method according to claim 1, is characterized in that: described polyadenosine is 12 ~ 20 adenosines.
3. method according to claim 1, is characterized in that: the length of step (1) described flag sequence is not less than 6bp.
4. method according to claim 3, is characterized in that: the length of described flag sequence is 9bp.
5. method according to claim 1, is characterized in that: in step (2), and the fluorescence molecule that fluorescent mark described in a step uses is 7-aminocoumarin-3-carbonic acid Acibenzolar, 7-diethylin tonka bean camphor-3-carbonic acid Acibenzolar, 7-aminocoumarin-3-carbonic acid Acibenzolar, ayapanin-3-carbonic acid Acibenzolar, 5/6-fluorescein carbonic acid Acibenzolar, fluorescein-5-lsothiocyanates (FITC), fluorescein-5 maleimide, 5-iodacetyl amido fluorescein, rhodamine 6G-5/6-carbonic acid Acibenzolar, Sulforhodamine B SULPHURYL CHLORIDE, X-rhodamine-6-carbonic acid Acibenzolar, Sulforhodamine 101 SULPHURYL CHLORIDE, four ethylamino rhodamine-5/6-carbonic acid Acibenzolars, four methylamino-rhodamine-6-maleimides, Dallas Pink C2 maleimide, four methylamino-rhodamine-5/6-lsothiocyanates, four methylamino-rhodamine-5/6-maleimides, dansyl chloride, EDANSC2 maleimide, iodacetyl amido EDANS, NBD-X Acibenzolar, ThioGlo-1 or CHROMISL5HNazide.
6. method according to claim 1, is characterized in that: in step (2), and the method that in a step, top sequence is connected with initial building block is:
By top sequence amination, initial building block carboxyl, sulfydryl or alkynyl, react and get final product.
7. method according to claim 1, is characterized in that: in step (2), and building-up process pH is 8 ~ 12, and temperature is 0 ~ 30 DEG C.
8. method according to claim 1, it is characterized in that: in step (2), between top sequence and flag sequence, flag sequence or the method for attachment of flag sequence and end sequence be: make 5 ' of single stranded DNA with many nucleoside kinases-hold phosphorylation, connect by RNA ligase.
9. method according to claim 8, is characterized in that: described many nucleoside kinases are the many nucleoside kinases of T4, and described RNA ligase is T4RNA ligase enzyme.
10. method according to claim 1, is characterized in that: step (3) described screening method is the screening method based on receptor-ligand specific reaction.
The synthesis of 11. 1 kinds of lead compounds and screening reagent box, is characterized in that: it comprises following composition:
1) i kind building block and (i+2) plant Single-stranded DNA fragments, and (i+2) plants Single-stranded DNA fragments and comprise i kind flag sequence, a kind of top sequence and a kind of end sequence, and i kind mark is sequence specific mark i kind building block respectively, wherein, i=1,2,3 ... n;
2) fluorescent mark reagent, top sequence are connected with initial building block and connect with reagent with reagent, combinational chemistry synthesis reagent and Single-stranded DNA fragments;
3) screening compound reagent;
4) DNA sequencing reagent;
Composition 1) 3 ' end connection ribonucleotide of described flag sequence; Described ribonucleotide is cytidine;
Composition 1) described top sequence comprises polyadenosine.
12. test kits according to claim 11, is characterized in that: described polyadenosine is 12 ~ 20 adenosines.
13. test kits according to claim 11, is characterized in that: composition 1) length of described flag sequence is not less than 6bp.
14. test kits according to claim 12, is characterized in that: the length of described flag sequence is 9bp.
15., according to test kit according to claim 11, is characterized in that: composition 2) described fluorescent mark reagent comprises fluorescence molecule, and described fluorescence molecule is 7-aminocoumarin-3-carbonic acid Acibenzolar, 7-diethylin tonka bean camphor-3-carbonic acid Acibenzolar, 7-aminocoumarin-3-carbonic acid Acibenzolar, ayapanin-3-carbonic acid Acibenzolar, 5/6-fluorescein carbonic acid Acibenzolar, fluorescein-5-lsothiocyanates (FITC), fluorescein-5 maleimide, 5-iodacetyl amido fluorescein, rhodamine 6G-5/6-carbonic acid Acibenzolar, Sulforhodamine B SULPHURYL CHLORIDE, X-rhodamine-6-carbonic acid Acibenzolar, Sulforhodamine 101 SULPHURYL CHLORIDE, four ethylamino rhodamine-5/6-carbonic acid Acibenzolars, four methylamino-rhodamine-6-maleimides, Dallas Pink C2 maleimide, four methylamino-rhodamine-5/6-lsothiocyanates, four methylamino-rhodamine-5/6-maleimides, dansyl chloride, EDANSC2 maleimide, iodacetyl amido EDANS, NBD-X Acibenzolar, ThioGlo-1 or CHROMISL5HNazide.
16. test kits according to claim 11, is characterized in that: composition 2) described top sequence is connected with building block by pack containing the reagent of amination single stranded DNA and the reagent of carboxyl, sulfydryl or alkynyl building block.
17. test kits according to claim 11, is characterized in that: composition 2) described Single-stranded DNA fragments connection reagent comprises multinuclear glycosides kinases and RNA ligase.
18. test kits according to claim 17, is characterized in that: described many nucleoside kinases are the many nucleoside kinases of T4, and described RNA ligase is that T4RNA ligase enzyme connects.
19. 1 kinds of combinatorial chemistry libraries, it is characterized in that: it take building block as raw material, with the combinatorial chemistry library of the combinational chemistry synthesis described in claim 1 ~ 10 any one, wherein, each compound label one section of single-stranded DNA sequence and a fluorescence molecule, the structure of this single-stranded DNA sequence is top sequence---i kind flag sequence---end sequence, the building block used in described i kind flag sequence specific mark i kind combinatorial chemistry building-up process, it puts in order identical with the addition sequence of building block in combinatorial chemistry building-up process.
20. combinatorial chemistry libraries according to claim 19, is characterized in that: the length of described flag sequence is not less than 6bp.
21. combinatorial chemistry libraries according to claim 20, is characterized in that: the length of described flag sequence is 9 bp.
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| US7377894B2 (en) * | 1999-09-29 | 2008-05-27 | Pharmacia & Upjohn Company | Methods for creating a compound library and identifying lead chemical templates and ligands for target molecules |
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