CN116121881A

CN116121881A - Synthesis method of double pharmacophore DNA coding compound library

Info

Publication number: CN116121881A
Application number: CN202211556531.XA
Authority: CN
Inventors: 李亦舟; 赵桂贤; 钟书婷; 张功
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2022-12-06
Filing date: 2022-12-06
Publication date: 2023-05-16

Abstract

The invention relates to the technical field of DNA coding compound libraries, in particular to a synthesis method of a double-pharmacophore DNA coding compound library, which constructs a reversible covalent Y-shaped DNA initial fragment and synthesizes the double-pharmacophore DNA coding compound library; performing controllable conversion of the double-pharmacophore DNA coding compound library and the single-pharmacophore DNA coding compound library; making the DNA encoding compound library compatible with a variety of screening systems; constructing a single pharmacophore DNA coding compound library into a dynamic compound library, and balancing and locking a dynamic structure by utilizing a covalent crosslinking group; combining the single-pharmacophore DNA coding compound library with other single-pharmacophore compound libraries to construct a self-assembled multi-pharmacophore library; the single pharmacophore DNA encoding compound pool was paired with the functional DNA conjugate by DNA hybridization and covalent cross-linking. The method of the invention improves the high efficiency of synthesis and the flexibility of screening of the DNA coding compound library through reversible covalent Y-shaped DNA initial fragments.

Description

Synthesis method of double pharmacophore DNA coding compound library

Technical Field

The invention relates to the technical field of DNA coding compound libraries, in particular to a synthesis method of a double pharmacophore DNA coding compound library.

Background

In the field of new drug development, high-throughput screening for biological targets is one of the main means for rapidly obtaining lead compounds. However, conventional high throughput screening based on single molecules requires long time, huge equipment investment, limited numbers of library compounds (millions), and the build-up of compound libraries requires decades of accumulation, limiting the efficiency and possibilities of discovery of lead compounds. Sydney Brenner and Richard Lerner teachings of the American Scripps institute in 1992 proposed the concept of a pool of DNA-encoding compounds (DNA Encoded Library, simply DEL) by linking an organic small molecule reagent to a unique sequence of DNA at the molecular level (i.e., DNA labeling the small molecule reagent), using a combinatorial chemistry "combinatorial-resolution" strategy to rapidly construct a huge number of libraries of compounds each consisting of different organic small molecule reagent residues and having a corresponding unique base sequence of DNA. The DNA coding compound library combines the combined chemistry and molecular biology technologies, each compound is added with a DNA label on the molecular level, the compound library of up to hundred million levels can be synthesized in extremely short time, the compound can be identified by a gene sequencing method, the size and the synthesis efficiency of the compound library are greatly increased, and the compound library becomes the trend of the next generation compound library screening technology. DNA-encoded compound library technology is beginning to be widely used in the pharmaceutical industry and has yielded a number of positive effects (Accounts ofChemical Research,2014,47,1247-1255).

In the development of drug screening technology for DNA coding compound libraries, synthesis and screening of DNA coding compound libraries are two key points, and often determine the success of drug screening. It is generally known that the higher the complexity of a library of compounds, i.e. the number of different structural motifs, chemical structures present, the greater the likelihood that a molecule with the activity of interest will be found by the library of compounds. Thus, in the synthesis of compound libraries, glaxoSmithKline (formerly Praecis) company or the like uses a head end (head piece) with a stable covalent hairpin structure as a starting fragment, and combines a grouping-merging-grouping (split-split) strategy to realize efficient coding and synthesis of compound libraries; the Neri team developed a technology of DNA encoded self-assembling chemical (ESAC) that based on complementary self-assembly of DNA strands between two or three single-stranded DNA encoded compound libraries, achieved rapid amplification of chemical structure and chemical diversity of the compound libraries, and achieved affinity maturation, expanded screening targets range, etc. However, because the synthesis of the single-stranded DNA coding compound library in ESAC technology can introduce auxiliary DNA reagent chains to increase the cost and complexity, the stability of a non-covalent binding system is low, DNA mismatch can occur, and information transfer operation is needed to carry out decoding operation and other limitations, the efficient synthesis technology for solving the double-pharmacophore DNA coding compound library has important technical and application significance.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for synthesizing a double pharmacophore DNA coding compound library, which improves the efficiency of synthesis and the flexibility of screening of the DNA coding compound library by constructing reversible covalent Y-type DNA initial fragments.

The invention solves the technical problems by the following technical means:

the invention discloses a synthesis method of a double pharmacophore DNA coding compound library, which comprises the following steps:

constructing reversible covalent Y-type DNA initial fragments to form orthogonal codes of DNA coding fragments with different sticky ends, and synthesizing a double-pharmacophore DNA coding compound library;

performing controllable conversion of the double-pharmacophore DNA coding compound library and the single-pharmacophore DNA coding compound library, and realizing effective decoding of the double-pharmacophore DNA coding compound library and the single-pharmacophore DNA coding compound library;

the DNA coding compound library is compatible with a plurality of screening systems, the target screening range of the coding compound library is enlarged, and the screening of the coding compound library comprises double pharmacophore DNA coding compound library screening and single pharmacophore DNA coding compound library screening;

dynamically combining single pharmacophore DNA coding compound libraries to construct dynamic DNA coding compound libraries, and balancing and locking dynamic structures by utilizing covalent reversible crosslinking groups;

Combining the single-pharmacophore DNA coding compound library with other single-pharmacophore compound libraries to construct a self-assembled multi-pharmacophore library;

through DNA hybridization and covalent cross-linking, the single pharmacophore DNA encoding compound library is combined with the functional compound DNA conjugate, so that the multifunction of the DNA encoding compound library is realized.

In some alternative embodiments, the construction of a reversible covalent "Y-type" DNA starting fragment comprises the steps of:

respectively synthesizing three single-stranded DNAs with X crosslinking groups, wherein the three single-stranded DNAs are single-stranded ssDNA-T, single-stranded ssDNA-B and single-stranded ssDNA-R, and the X crosslinking groups are special crosslinking groups or natural bases;

construction strategy of the reversible covalent "Y-type" DNA initiation fragment:

forming reversible covalent Y-shaped DNA initial fragments by covalent crosslinking chemical reaction of two single-stranded DNAs containing special crosslinking groups and complementary single-stranded DNAs not containing the special crosslinking groups;

dissociating the "Y-type" DNA initial fragment formed by covalent cross-linking into a single-stranded initial state by covalent dissociation chemical reaction;

construction strategy two of the reversible covalent "Y-type" DNA initial fragment:

respectively synthesizing three single-stranded DNA with X crosslinking groups, wherein the X crosslinking groups are special crosslinking groups or natural bases;

Forming an intermediate DNA with a covalent structure by covalent crosslinking chemical reaction of a single-stranded DNA containing a special crosslinking group and a complementary single-stranded DNA not containing the special crosslinking group; then forming a reversible covalent Y-shaped DNA initial fragment by covalent crosslinking chemical reaction with another complementary single-stranded DNA containing special crosslinking base;

construction strategy three of the reversible covalent "Y-type" DNA initiation fragment:

three single-stranded DNA containing special crosslinking groups are subjected to covalent crosslinking chemical reaction to form a novel reversible covalent Y-shaped DNA initial fragment;

the construction cycle times of the reversible covalent Y-shaped DNA initial fragment are not less than 50 times;

the reversible covalent Y-type DNA initial fragment comprises at least two reaction sites R for synthesizing a compound library, and a connector L is connected between the reaction sites R and the single-stranded DNA.

In some alternative embodiments, the single-stranded ssDNA-T, single-stranded ssDNA-B, and single-stranded ssDNA-R each have a complementary region therebetween, each of the complementary regions having a length of 0 to 30 bases, and at least one of the single-stranded ssDNA-T, single-stranded ssDNA-B, and single-stranded ssDNA-R comprises at least one X crosslinking group;

the X crosslinking group is positioned on a reversible covalent Y-type DNA initial fragment, and is any one of the following special crosslinking groups 1-12:

the natural base is any one of adenine, guanine, cytosine and thymine.

In some alternative embodiments, the controllable transformation of the dual pharmacophore DNA encoding compound library with the single pharmacophore DNA encoding compound library comprises the steps of:

the synthesis of a DNA coding compound library is realized by adopting a split-pool-split strategy;

carrying out DNA compatible chemical reaction on reversible covalent Y-shaped DNA initial fragments and chemical structural motifs, wherein the chemical structural motifs are any one of carboxylic acid, aldehyde, olefin, amine, boric acid and halide;

adding the coding DNA label corresponding to each chemical structural element, and connecting the DNA cohesive ends through a DNA ligase or a chemical method; or adding a chemical structural element into two chemical reaction sites of the 'Y-shaped' DNA initial fragment, adding the corresponding coded DNA labels with different sticky ends together, and connecting the sticky ends of the DNA by a DNA ligase or a chemical method;

The connection sequence of the DNA label and the chemical structural element in each dimension can be exchanged, and the samples in the same dimension are uniformly mixed by adopting a combination chemical mode, so that the next dimension synthesis is performed;

after the synthesis of the double-pharmacophore DNA coding compound library is completed, a covalent bond is broken to open a special Y-type structure, and a selective degradation condition is used for degrading or separating a DNA chain which is not connected with the coding compound in the library establishment process, so that the conversion of the double-pharmacophore DNA coding compound library and the single-pharmacophore DNA coding compound library is completed.

In some alternative embodiments, the dual pharmacophore DNA encoding compound library screening comprises the steps of:

the double pharmacophore DNA encoding compound library screening strategy is:

mixing known active compounds into any single pharmacophore compound library to synthesize a double pharmacophore DNA coding compound library;

incubating and screening the double pharmacophore DNA encoding compound library and related targets of the active compounds;

the members of the library of compounds that do not bind to the target or have weak affinity are separated by selective degradation conditions, and the members of the library of compounds that have stronger affinity to the target are enriched;

structural information of the enriched library members is decoded by polymerase chain reaction and DNA sequencing techniques.

The screening strategy of the double pharmacophore DNA coding compound library is II:

using the protein target as a screening substrate of a double pharmacophore DNA coding compound library;

incubating and screening the double pharmacophore DNA encoding compound library with a target;

the members of the library of compounds that do not bind to the target or have a weak affinity are separated by selective degradation conditions and the members of the library of compounds that have a strong affinity to the target are enriched;

In some alternative embodiments, the single pharmacophore DNA encoding compound library screening further comprises the steps of:

screening strategy one of the single pharmacophore DNA coding compound library:

hybridizing a single pharmacophore DNA encoding compound library with a DNA probe having chemical reactivity;

when a compound with affinity in the DNA coding compound library is combined with a target, the distance between the DNA probe and the target is shortened, and covalent crosslinking between the DNA probe and the target is promoted;

separating the compound DNA conjugate which is not bound with the target through selective degradation conditions, and enriching the DNA compound conjugate which is bound with the target;

Amplification by polymerase chain reaction and decoding of compound structural information by DNA sequencing technology;

the single pharmacophore DNA encoding compound library screening strategy two:

incubating a single pharmacophore DNA encoding compound library with a target connected with a PCR primer sequence DNA, and carrying out PCR amplification on a hybridization structure of a compound library member with affinity to the target and the DNA primer sequence connected with the target to obtain structural information of a compound with high affinity to the target;

the single pharmacophore DNA encoding compound library screening strategy three:

hybridizing and covalently crosslinking a single pharmacophore DNA encoding compound library with a DNA probe having chemical reactivity;

when a compound with affinity in the compound library is combined with a target, the distance between the DNA probe and the target is shortened, the DNA probe and the target are promoted to carry out covalent crosslinking, and a covalent crosslinking compound of single-stranded DNA connected with the information of the compound library, the DNA probe with chemical reactivity and the target is realized;

and (3) separating out the three covalent crosslinking complexes by utilizing a target in-vitro binding experiment, and further obtaining structural information of a compound with high affinity with the target through DNA decoding.

In some alternative embodiments, the selective degradation condition is any one of exonuclease, chemical reaction condition, separation capture, and the exonuclease is any one of ExoI, exoIII, λexo.

In some alternative embodiments, the construction of the dynamic combinatorial compound library further comprises the steps of:

converting the double-pharmacophore DNA coding compound library into a single-pharmacophore DNA coding compound sub-library A and a single-pharmacophore DNA coding compound sub-library B, and respectively dynamically combining with a corresponding single-strand DNA coding compound sub-library C or sub-library D;

under the promotion of the target, the DNA conjugates of the compounds with affinity in the two compound libraries are close to each other, so that hybridization of DNA complementary regions is promoted;

covalent crosslinking is carried out on the hybridized double-stranded DNA by utilizing the crosslinking characteristic of the special crosslinking group X, so that the combination of specific target compounds is stabilized;

the coding information of double-stranded DNA is integrated into the same single-stranded DNA, and the structure information of the enriched compound is decoded by PCR amplification, DNA sequencing and the like.

In some alternative embodiments, the construction of the self-assembled multi-pharmacophore pool further comprises the steps of:

the single pharmacophore DNA codes a compound library through self-assembly, and the structure of the self-assembled DNA is stabilized through a special crosslinking group X;

the single-stranded DNA encoding compound sub-pool A, B, C is hybridized to form a double-pharmacophore or triple-pharmacophore compound pool, and the compound pool size is enlarged.

In some alternative embodiments, the multifunctionalization of the DNA encoding compound library further comprises the steps of:

Converting the double-pharmacophore DNA coding compound library into a single-pharmacophore DNA coding compound library and hybridizing with single-stranded DNA connected with different functional compounds;

and then a stable hybridization structure is formed by utilizing a special crosslinking group X, so that the multifunction of the DNA coding compound library is realized.

The invention adopting the technical scheme has the following beneficial effects:

(1) According to the synthesis method of the double-pharmacophore DNA coding compound library, a special reversible covalent Y-type DNA (oligonucleotide) initial fragment is constructed, and the Y-type DNA coding compound library with a bifurcated coding region is constructed, so that compared with the traditional DNA coding compound library with a linear coding region, the synthesis limitation of the single-pharmacophore DNA coding compound library can be broken, and meanwhile, the chemical information of a plurality of sub-libraries is coded, so that the breakthrough of the convenient synthesis technology of the double-pharmacophore DNA coding compound library is realized;

(2) The synthesis method of the Y-type double-pharmacophore DNA coding compound library can rapidly expand the scale and diversity of the compound library under the condition of ensuring the high efficiency of coding in the form of forked coding DNA;

(3) Compared with the traditional self-assembled compound library synthesis strategy, the synthesis method of the Y-type double-pharmacophore DNA coding compound library does not need to independently synthesize sub-libraries, does not need to carry out recombination among the compound libraries, and is more convenient and efficient in DNA ligase-mediated connection of double-stranded DNA adhesive ends than that of a single-stranded DNA splint method;

(4) The synthesis method of the Y-type double-pharmacophore DNA coding compound library can directly adopt commercial coding DNA, and is more suitable for industrial application;

(5) Compared with the traditional self-assembled compound library, the synthesis method of the Y-type double-pharmacophore DNA coding compound library has shorter DNA chain length, and is more beneficial to reducing the influence of a coding region on ligand binding;

(6) The double pharmacophore compound library constructed by the synthesis method of the 'Y-type' double pharmacophore DNA coding compound library can be used for screening affinity maturation and other applications;

(7) Based on the design of reversible covalent 'Y-type' DNA (oligonucleotide) initial fragments, the strategy can realize reversible interconversion between a double-pharmacophore DNA coding compound library and a single-pharmacophore DNA coding compound library;

(8) After the double-pharmacophore DNA coding compound library is converted into the single-pharmacophore DNA coding compound library, the functions of compound library screening, compound library detection, compound library delivery and the like can be realized through crosslinking with the DNA connected with the functional compound.

Drawings

FIG. 1 is a schematic representation of a reversible covalent "Y-type" DNA starting fragment of the present invention;

FIG. 2 is a representative structure of a particular crosslinking group X of the present invention;

FIG. 3 is a schematic diagram of reversible covalent crosslinking and dissociation of the present invention;

FIG. 4 is a schematic diagram showing the construction of a library of DNA encoding compounds of the present invention;

FIG. 5 is a schematic representation of the conversion of a double-pharmacophore DNA encoding compound pool of the invention into a single-pharmacophore DNA encoding compound pool;

FIG. 6 shows a screening strategy I compatible with the double pharmacophore DNA coding system of the present invention;

FIG. 7 is a second screening strategy compatible with the double pharmacophore DNA coding system of the present invention;

FIG. 8 is a screen strategy I compatible with the single pharmacophore DNA coding system of the present invention;

FIG. 9 is a second screening strategy compatible with the single pharmacophore DNA coding system of the present invention;

FIG. 10 is a screen strategy III compatible with the single pharmacophore DNA coding system of the present invention;

FIG. 11 is a schematic diagram showing the compatibility of the DNA coding system of the present invention with a dynamic library;

FIG. 12 is a schematic diagram of the compatibility of the DNA coding system of the present invention with self-assembled multiple pharmacophore libraries;

FIG. 13 is a schematic representation of the functionalization of a library of DNA encoding compounds of the present invention;

FIG. 14 is a DNA cross-linking chromatogram of the present invention;

FIG. 15 is a chromatogram of repeated crosslinking dissociation of the DNA of the present invention;

FIG. 16 shows the sequencing results of the present invention;

FIG. 17 is a dynamic combinatorial compound library verification of the invention;

fig. 18 is a schematic diagram of the invention suitable for cell delivery and fluorescence imaging.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The embodiment of the invention provides a synthesis method of a double-pharmacophore DNA coding compound library, which comprises the following steps:

Wherein, the construction of the reversible covalent "Y-type" DNA initial fragment comprises the following steps:

three single-stranded DNAs with X crosslinking groups are respectively synthesized, wherein the three single-stranded DNAs are single-stranded ssDNA-T, single-stranded ssDNA-B and single-stranded ssDNA-R, and at least one single-stranded DNA of the single-stranded ssDNA-T, the single-stranded ssDNA-B and the single-stranded ssDNA-R contains at least one X crosslinking group. Wherein ssDNA-T and ssDNA-B have complementary regions 1, ssDNA-T and ssDNA-R have complementary regions 2, and ssDNA-B and ssDNA-R have complementary regions 3. Each segment of the complementary region is between 0 and 30 bases in length, and the complementary region length is not required to be uniform. X crosslinking groups are special crosslinking groups or natural bases, and are positioned on reversible covalent Y-type DNA initial fragments, but are not fixed at a specific site. X crosslinking group is any one of special crosslinking groups 1-12 shown in figure 2, and natural base is any one of adenine, guanine, cytosine and thymine

Construction strategy of reversible covalent "Y-type" DNA initiation fragment:

forming reversible covalent Y-shaped DNA initial fragments by covalent crosslinking chemical reaction of two single-stranded DNAs containing special crosslinking groups and complementary single-stranded DNAs not containing the special crosslinking groups; the "Y-type" DNA starting fragment formed by covalent cross-linking is dissociated into a single-stranded initial state by covalent dissociation chemical reaction.

Construction strategy of reversible covalent "Y-type" DNA initiation fragment two:

respectively synthesizing three single-stranded DNA with X crosslinking groups, wherein the X crosslinking groups are special crosslinking groups or natural bases; forming an intermediate DNA with a covalent structure by covalent crosslinking chemical reaction of a single-stranded DNA containing a special crosslinking group and a complementary single-stranded DNA not containing the special crosslinking group; then forming a reversible covalent Y-shaped DNA initial fragment by covalent crosslinking chemical reaction with another complementary single-stranded DNA containing special crosslinking base; the "Y-type" DNA starting fragment formed by covalent cross-linking is dissociated into a single-stranded initial state by covalent dissociation chemical reaction.

Construction strategy three of reversible covalent "Y-type" DNA initiation fragment:

respectively synthesizing three single-stranded DNA with X crosslinking groups, wherein the X crosslinking groups are special crosslinking groups or natural bases; three single-stranded DNA containing special crosslinking groups are subjected to covalent crosslinking chemical reaction to form a novel reversible covalent Y-shaped DNA initial fragment; the "Y-type" DNA starting fragment formed by covalent cross-linking is dissociated into a single-stranded initial state by covalent dissociation chemical reaction.

The construction cycle times of the reversible covalent Y-type DNA initial fragment are not less than 50 times;

the reversible covalent Y-type DNA initial fragment comprises at least two reaction sites R for synthesizing a compound library, wherein the reaction sites R1 and R2 are respectively, a connector L is connected between the reaction sites R and the single-stranded DNA, and the connector L comprises a connector L1 and a connector L2.

Wherein the controllable transformation of the double pharmacophore DNA encoding compound library and the single pharmacophore DNA encoding compound library comprises the following steps:

the synthesis of a DNA coding compound library is realized by adopting a split-pool-split strategy; carrying out DNA compatible chemical reaction on reversible covalent Y-type DNA initial fragments and chemical structural motifs, wherein the chemical structural motifs are any one of carboxylic acid, aldehyde, olefin, amine, boric acid and halide; adding the coding DNA label corresponding to each chemical structural element, and connecting the DNA cohesive ends through a DNA ligase or a chemical method; or adding a chemical structural element into two chemical reaction sites of the 'Y-shaped' DNA initial fragment, adding the corresponding coded DNA labels with different sticky ends together, and connecting the sticky ends of the DNA by a DNA ligase or a chemical method; the connection sequence of the DNA label and the chemical structural element in each dimension can be exchanged, and the samples in the same dimension are uniformly mixed by adopting a combination chemical mode, so that the next dimension synthesis is performed; after the synthesis of the double-pharmacophore DNA coding compound library is completed, a covalent bond is broken to open a special Y-type structure, and a selective degradation condition is used for degrading or separating a DNA chain which is not connected with the coding compound in the library establishment process, so that the conversion of the double-pharmacophore DNA coding compound library and the single-pharmacophore DNA coding compound library is completed.

Wherein, the screening of the double pharmacophore DNA coding compound library comprises the following steps:

screening strategy of double pharmacophore DNA coding compound library: mixing known active compounds into any single pharmacophore compound library to synthesize a double pharmacophore DNA coding compound library; incubating and screening the double pharmacophore DNA encoding compound library and related targets of the active compounds; the members of the library of compounds that do not bind to the target or have weak affinity are separated by selective degradation conditions, and the members of the library of compounds that have stronger affinity to the target are enriched; structural information of the enriched library members is decoded by polymerase chain reaction and DNA sequencing techniques.

Screening strategy two of double pharmacophore DNA coding compound library: using the protein target as a screening substrate of a double pharmacophore DNA coding compound library; incubating and screening the double pharmacophore DNA encoding compound library with a target; the members of the library of compounds that do not bind to the target or have a weak affinity are separated by selective degradation conditions and the members of the library of compounds that have a strong affinity to the target are enriched; structural information of the enriched library members is decoded by polymerase chain reaction and DNA sequencing techniques.

Wherein, the screening of the single pharmacophore DNA coding compound library further comprises the following steps:

single pharmacophore DNA encoding compound library screening strategy one:

hybridizing a single pharmacophore DNA encoding compound library with a DNA probe having chemical reactivity; when a compound with affinity in the DNA coding compound library is combined with a target, the distance between the DNA probe and the target is shortened, and covalent crosslinking between the DNA probe and the target is promoted; separating the compound DNA conjugate which is not bound with the target through selective degradation conditions, and enriching the DNA compound conjugate which is bound with the target; amplification by polymerase chain reaction and decoding of compound structural information by DNA sequencing technology;

single pharmacophore DNA encoding compound library screening strategy two:

single pharmacophore DNA encoding compound library screening strategy three:

hybridizing and covalently crosslinking a single pharmacophore DNA encoding compound library with a DNA probe having chemical reactivity; when a compound with affinity in the compound library is combined with a target, the distance between the DNA probe and the target is shortened, the DNA probe and the target are promoted to carry out covalent crosslinking, and a covalent crosslinking compound of single-stranded DNA connected with the information of the compound library, the DNA probe with chemical reactivity and the target is realized; and (3) separating out the three covalent crosslinking complexes by utilizing a target in-vitro binding experiment, and further obtaining structural information of a compound with high affinity with the target through DNA decoding.

The selective degradation condition is any one of exonuclease, chemical reaction condition and separation capture, and the exonuclease is any one of ExoI, exoIII and lambda Exo.

Example 1

Construction of reversible covalent "Y-type" DNA (oligonucleotide) initiation fragment:

as shown in fig. 1 and 2, covalent reversible chemical reaction is adopted to realize efficient reversible covalent cross-linking between double-stranded DNA strands, so as to form a reversible covalent hairpin structure. Wherein, the

special crosslinking groups

1, 2, 4 and 7 can generate cycloaddition crosslinking reaction under the illumination of ultraviolet wavelength in the range of 200-400nm, and the crosslinking products can generate cyclocracking reaction under the illumination of corresponding different ultraviolet wavelengths; the special crosslinking group 5 can react under the oxidation reaction condition (such as potassium carbonate, sodium hydroxide, bismuth nitrate catalytic oxidation condition and the like) for generating S-S bond to generate S-S crosslinking product, and the crosslinking product can simultaneously undergo cleavage reduction reaction under the reduction reaction condition (such as beta-mercaptoethanol (beta-ME), dithiothreitol (DTT) and the like) for disulfide bond. The special crosslinking group 6 can react under free radical oxidation conditions (such as photocatalysis, high-energy radiation and the like) to generate cycloaddition crosslinking products, and the crosslinking products can also undergo reduction reaction under reduction reaction conditions. As shown in fig. 14, this procedure was confirmed by high performance liquid chromatography (High Performance Liquid Chromatography, HPLC).

Example 2:

as shown in fig. 1 and 3, two single-stranded DNAs each containing a special crosslinking group 3-cyanovinylcarbazole and the complementary single-stranded DNA are subjected to cycloaddition reaction between the special base 3-cyanovinylcarbazole and a T base or a C base obliquely aligned on the complementary DNA under the illumination of 340-380nm to form a stable covalent bond, so that covalent crosslinking among three single-stranded DNA strands is realized; under the illumination of 300-320nm, the cycloaddition reaction is reduced to an initial state before the cycloaddition reaction occurs. As shown in FIG. 15, the process is detected by HPLC, and the result shows that the strategy can realize multiple interconversions of single-stranded DNA and double-stranded DNA, thereby greatly promoting the flexibility of DNA coding compound libraries.

Example 3

By adopting the construction method, the DNA crosslinking and dissociation process can be realized for more than 50 times without damaging the DNA.

Example 4

After validating the synthesis of the reversibly cross-linked "Y-type" DNA (oligonucleotide) starting fragment, a library of DNA-encoding polypeptide compounds is further synthesized. The sequences and the corresponding structural group types corresponding to the two dimensions are (151 types and 95 types) x (156 types and 97 types). According to the synthesis method shown in fig. 4 to 5, a 1mM concentration of the starting DNA fragment solution was dispensed into 381 consecutive wells of a 96-well plate, 10 μl of each well was continuously added with 11 μl of each of the upper and lower strands of the first cycle of labeled nucleotide double strand having a concentration of 1mM, DNA tag ligation was performed, ethanol precipitation purification was performed, the purified DNA was continuously dissolved in 10 μl of sodium borate buffer solution (ph=9.5, 250 mM), the corresponding compound structural unit was further added, after the reaction was completed, all the reaction solutions were mixed, ethanol precipitation was performed again, and the resulting DNA precipitate was dissolved in 200 μl double distilled water, and the product was desalted and purified by using a 10K size ultrafiltration tube having a size of 500 μl to obtain the first cycle of product. The latter two cycles used the same "split-pool-split" to synthesize a pool of 151 x 95 x 156 x 97 = 217,068,540 compounds encoded by DNA. After synthesis of the compound library, it was subjected to single-chain compound library conversion and characterized by liquid chromatograph-mass spectrometer (liquid chromatograph-mass spectrometer, LC-MS).

Example 5: screening of double pharmacophore DNA encoding Compound libraries

Screening according to the method shown in fig. 6 by using carbonic anhydrase IX (Carbonic Anhydrase IX, CA IX) as a target protein, purifying the double-pharmacophore DNA coding compound library constructed in examples 1-5 (wherein a known affinity molecular structure of the target protein is contained in one pharmacophore library) by using DNA selective degradation conditions (the DNA of an unconnected compound is degraded by lambda Exo), removing members which are not bound or weakly bound with the target protein (CA IX) after incubating for 8 hours in a buffer system, enriching high affinity library members of the target protein, and performing structural decoding on the screened and enriched compound by PCR amplification and DNA sequencing to realize affinity maturation research of the known affinity molecules of the target protein.

The library of double pharmacophore DNA encoding compounds constructed in examples 1-5 was purified by DNA selective degradation conditions (λExo degrading the DNA of the unligated compounds) using murine sarcoma virus protein (kirsten rat sarcoma viral oncogene, KRAS) as the target protein, screening as shown in FIG. 7, and after incubation with the target protein (KRAS) in a buffer system for 8 hours, the members that did not bind or did not bind weakly were removed and the members of the high affinity library of target proteins were enriched. And then the structure decoding is carried out on the screened compound through PCR amplification and DNA sequencing, and the high affinity binding molecule of the target protein with large surface area is found.

As shown in fig. 16, sequencing data clearly shows that the mixed sequences and expectations were identical prior to screening; but clearly revealed that the specific sequence (DE-2-493) was enriched after the screening was completed. The data prove that the coding technology using reversible covalent Y-type hairpin structure DNA as an initial fragment can be used for library construction and screening, and the efficient synthesis and the diversified application of the double-pharmacophore DNA coding compound library are truly realized. It should be noted that in practical application, the coding dimension is not limited to the four dimensions in the above examples, so that a compound library with larger specification can be constructed; the variety of the library is not limited to the construction of the polypeptide library, so that a compound library with higher chemical diversity can be constructed; the targets screened are not limited to proteins, and thus can be screened against other targets.

Example 6: screening of single pharmacophore DNA encoding libraries of compounds

The double pharmacophore DNA encoding compound libraries constructed in examples 1-5 were transformed into single pharmacophore DNA encoding compound libraries by DNA selective degradation conditions (degradation of the DNA of the unligated compounds by λExo) using α1-Acid Glycoprotein (AGP) as a protein target, and screening as shown in FIG. 8. Hybridization is then carried out with probes having chemically reactive groups (biaziridines), the target protein (AGP) is added, incubation is carried out in a buffer system for 8 hours, covalent crosslinking of the protein is carried out by irradiation of the active probes with ultraviolet radiation at 365 nm. And then through a special crosslinking group 7 in DNA: 3-cyanoethylene carbazole, and the two DNA strips are subjected to photocrosslinking under the irradiation of ultraviolet wavelength of 360-400nm to form a stable double-chain structure. And finally, degrading the DNA label of the compound DNA conjugate which does not have binding force with the target protein in the system by adding exonuclease ExoI. The screened compounds were then structurally decoded by PCR amplification and DNA sequencing.

Screening was performed according to the method shown in fig. 9, the target protein was chemically linked to DNA with a primer sequence, incubated in a buffer solution for 8 hours with a library of encoded compounds that were converted to single pharmacophore DNA, and then analyzed by PCR techniques, and for compounds that have binding capacity to the target protein, the encoded DNA could be amplified by PCR for sequencing and decoding.

As shown in fig. 16, sequencing data clearly shows that the mixed sequences and expectations were identical prior to screening; but after the screening was completed, clearly the specific sequence (DE-3-117) was enriched. The data prove that the coding technology using reversible covalent Y-type hairpin structure DNA as an initial fragment can be used for library construction and screening, and the interconversion of the double-pharmacophore DNA coding compound library and the single-pharmacophore DNA coding compound library is truly realized. It should be noted that in practical application, the coding dimension is not limited to the four dimensions in the above examples, so that a compound library with larger specification can be constructed; the variety of the library is not limited to the construction of the polypeptide library, so that a compound library with higher chemical diversity can be constructed; the targets screened are not limited to proteins, and thus can be screened against other targets.

Example 7: construction of diverse compound libraries

As shown in FIGS. 6 to 10, two single-stranded DNAs each comprising a short complementary region and having a Desthiobiotin (DB) group attached thereto were prepared as ligands, and were verified by gel electrophoresis using streptavidin as a target protein, as shown in FIG. 17. At 0℃the two single stranded DNA forms a stable double strand and under crosslinking conditions (I') a large amount of crosslinked double strand product is visible. In contrast, double strand was dynamic at 30℃and no crosslinking (I) was observed. By adding the target protein streptavidin, the cross-linked product becomes visible (II), indicating that binding of the target ligand promotes DNA hybridization and thus inter-strand cross-linking of double stranded DNA. The control experiments used the non-target protein bovine serum albumin or DNA without DB ligand, which showed no cross-linked products (III and IV). Indicating that the crosslinking group X is capable of specifically crosslinking the target to promote formation of a DNA duplex. The coding strategy is therefore compatible with dynamic combinatorial libraries, strongly facilitating rapid construction of libraries of compounds with higher chemical diversity.

Example 8

After conversion of the double-pharmacophore DNA-encoding compound pool into a single-pharmacophore DNA-encoding compound pool, it is incubated with single-stranded DNA with cell penetrating peptide attached to a fluorophore (fluorescein) through special cross-linked bases 7 in the DNA on the compound DNA conjugate: 3-cyanovinylcarbazole and single-stranded DNA linked with cell penetrating peptide form covalent crosslinking between DNA chains under the irradiation of ultraviolet wavelength of 360-400 nm. The cell permeability experiment was performed by selecting normal human mammary cells, and observing the transmembrane effect of the DNA encoding compound library by fluorescence confocal microscopy, as shown in FIG. 18, demonstrated that functionalization of the DNA encoding compound library can be achieved by hybridization with a compound having a functional group.

The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention. The technology, shape, and construction parts of the present invention, which are not described in detail, are known in the art.

Claims

1. A method for synthesizing a double pharmacophore DNA encoding compound library, which is characterized by comprising the following steps:

2. The method of claim 1, wherein said constructing a reversible covalent "Y-type" DNA starting fragment comprises the steps of:

3. The method for synthesizing a library of double pharmacophore DNA encoding compounds according to claim 2 wherein said single stranded ssDNA-T, single stranded ssDNA-B and single stranded ssDNA-R each have complementary regions therebetween, each of said complementary regions being between 0 and 30 bases in length, at least one of said single stranded ssDNA-T, single stranded ssDNA-B and single stranded ssDNA-R comprising at least one X crosslinking group;

the natural base is any one of adenine, guanine, cytosine and thymine.

4. A method of synthesizing a dual pharmacophore DNA encoding compound library according to claim 3 wherein said controllable conversion of said dual pharmacophore DNA encoding compound library to a single pharmacophore DNA encoding compound library comprises the steps of:

5. The method for synthesizing a double-pharmacophore DNA encoding compound library according to claim 4, wherein said double-pharmacophore DNA encoding compound library screening comprises the steps of:

the double pharmacophore DNA encoding compound library screening strategy is:

decoding structural information of the enriched compound library members by polymerase chain reaction and DNA sequencing technology;

6. The method of claim 5, wherein the single pharmacophore DNA encoding compound library screening further comprises the steps of:

screening strategy one of the single pharmacophore DNA coding compound library:

the single pharmacophore DNA encoding compound library screening strategy two:

7. The method for synthesizing a double-pharmacophore DNA coding compound library according to claim 6, wherein the selective degradation condition is any one of exonuclease, chemical reaction condition and separation capture, and the exonuclease is any one of ExoI, exoIII and lambda Exo.

8. The method for synthesizing a double pharmacophore DNA encoding compound library according to claim 7 wherein said dynamic combinatorial compound library construction further comprises the steps of:

9. The method for synthesizing a double-pharmacophore DNA encoding compound library according to claim 8 wherein said self-assembled multi-pharmacophore library construction further comprises the steps of:

10. The method of claim 9, wherein the multi-functionalization of the DNA encoding compound library further comprises the steps of: