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Methods for tagging dna-encoded libraries

A coding and library technology, applied in the direction of DNA preparation, recombinant DNA technology, chemical library, etc., can solve problems such as inability to shift

Active Publication Date: 2017-12-01
X CHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, most oligonucleotide linkage structures generated by chemical ligation reactions result in linkages that cannot be displaced by polymerases

Method used

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  • Methods for tagging dna-encoded libraries
  • Methods for tagging dna-encoded libraries
  • Methods for tagging dna-encoded libraries

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0206] Example 1. Preparation of components for chemical ligation (double-stranded head fragment and double-stranded label)

[0207] The head fragment HP006 chemically phosphorylated at the 5' end, SEQ ID NO: 1 -(p)CCTGTGTTZTTCACGGCCT, where Z represents C6-amino dT modification, was obtained from Biosearch Inc. HP006 was subsequently modified by DMT-MM acylation with Fmoc-NH-PEG4-CH2CH2COOH (Chem Pep Inc) using the following method.

[0208] 50 equivalents of Fmoc-NH-PEG4-CH2CH2COOH (Chem Pep Inc) were dissolved in DMA (dimethylacetamide, Acros), and 1 equivalent of HP006 dissolved in 0.5M borate buffer at pH 9.5 was added and 50 equivalents of DMT-MM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine hydrochloride, Acros) freshly dissolved in water . The reaction was allowed to proceed for 2-4 hours, followed by a second addition of 50 equiv of Fmoc-NH-PEG4-CH2CH2COOH and 50 equiv of DMT-MM, and then the reaction was allowed to proceed overnight. The reaction was mo...

Embodiment 2

[0214] Embodiment 2: chemical connection of double-stranded head fragment and double-stranded label

[0215] Fmoc-amino-PEG4-HP013 and double-stranded TagZA oligonucleotides were dissolved in 80 mM MES buffer (containing 800 mM NaCl and 8 mM ZnCl 2 ) to a final concentration of 0.33 mM. 1-cyanoimidazole was freshly dissolved in DMF at a concentration of 1 M, and 1-2 additions were made to the reaction over 12 hours to a final concentration of 1-cyanoimidazole of 150 mM. Reactions were then incubated overnight at 4°C.

[0216] Completed reactions were analyzed by denaturing gel electrophoresis and LCMS. Samples were then separated in a 15% denaturing analytical TBE-8M urea gel and visualized on TLC plates by UV shadowing using fluorescent dyes (254 nm). LCMS confirmed the formation of a double-stranded ligated product with MW 25,417.3 (calculated molecular weight 25,415.3 ) and ~70% conversion. Additional products of MW 20,254.7 and 18,935.4 were observed, corresponding to ...

Embodiment 3

[0225] Example 3. Fmoc deprotection of chemical ligation reaction products.

[0226]The product of the 1-cyanoimidazole ligation reaction was ethanol precipitated, dissolved in water and deprotected by incubation in 10% piperidine for 2 hours at room temperature. After this deprotection step, the material was purified on a 15% TBE-8M urea gel. LC-MS on the purified sample confirmed the presence of deprotected amino-PEG4-HP013-TagZA (MW 25,192.4, calculated molecular weight 25,193.2) and two semi-linked deprotected products (MW 18,738.6 and 20,029.3).

[0227] Integration of the LC traces gave a relative yield of 64% for the full-length product and about 18% for each of the hemiligated products. The ligation efficiency of each strand was predicted to be 83%.

[0228] Deprotection of the amino group by piperidine as Figure 4A shown. Gel purification of ligation reaction products: 15% TBE-urea gel, UV contrast such as Figure 4B shown. LCMS analysis of purified material as...

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Abstract

The present invention relates to methods for producing encoded chemical entities. In particular, the oligonucleotides and methods can include encoded chemical entities having wild-type linkages formed through chemical ligation techniques. One strategy that can be utilized that simultaneously takes advantage of chemical ligation as a means to encode chemical history, while also retaining the ability of polymerases to directly recover tag sequence and association information, is to perform chemical ligation in a manner that generates wildtype phosphodiester linkages. Such methods generally utilize condensing agents such as cyanogen bromide or similar along with 5'-phosphate and 3'-hydroxyl oligonucleotides in a double-stranded or templated context. Similarly cyanogen bromide has also been shown to chemically ligate pairs of substrate oligonucleotides that are 5'-hydroxyl and 3'-phosphate. However, these methods suffer from poor efficiency making them ill-suited for use in an iterative process such as tagging DNA-encoded libraries.

Description

Background of the invention [0001] DNA-encoded chemical library members are chemical entities generated by combinatorial chemical synthesis methods that are associated with combinations of encoding oligonucleotide tags. The combination of markers associated with individual library members can be determined and used to deduce the chemical synthesis history of the associated library member. [0002] One method for generating such libraries is a method in which oligonucleotide tags are serially chemically ligated to head fragment oligonucleotides by successive split-mix steps, thereby revealing a chemically generated entity. In each resolution step, a chemical synthesis step is performed together with an oligonucleotide ligation step. [0003] A chemically-mediated rather than an enzyme-mediated oligonucleotide ligation step allows greater flexibility with solution conditions and can reduce buffer exchange steps that may be necessary for thousands of small volumes of individual ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/02
CPCC07H21/02C07H1/00C12N15/1068C40B50/10C12N15/1065
Inventor A.D.基夫A.利托夫基克M.克拉克R.W.沃纳
Owner X CHEM
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