Systematic evolution of ligands by exponential enrichment: Chemi-SELEX

a technology of exponential enrichment and ligands, applied in the field of systematic evolution of ligands by exponential enrichment, can solve the problems of few successes and perform over a somewhat narrow range of chemical possibilities, and achieve the effect of less laborious

Inactive Publication Date: 2006-04-27
GILEAD SCI INC
View PDF17 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0016] In another embodiment, this invention provides a method for the subtractive separation of desirable ligands from less desirable ligands. This embodiment takes advantage of the strong interaction between the nucleic acid and / or it associated functional unit and the target to partition the covalently attached or strongly non-covalently attached nucleic acid-target complexes from free nucleic acids.
[0017] In another embodiment, subtractive separation is further exploited to automate the entire selection process. This embodiment makes the selection process much less labor intensive and provides the methods and apparatus to accomplish said automation.

Problems solved by technology

Despite intense recent investigation to identify RNA or DNA catalysts, few successes have been identified.
Among the drawbacks are that they act slowly relative to protein enzymes, and as described above, they perform over a somewhat narrow range of chemical possibilities.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Systematic evolution of ligands by exponential enrichment: Chemi-SELEX
  • Systematic evolution of ligands by exponential enrichment: Chemi-SELEX

Examples

Experimental program
Comparison scheme
Effect test

example 1

5′-phosphorothioate-modified RNA binding to N-bromoacetyl-bradykinin

[0063] This example describes a Chemi-SELEX procedure wherein RNA is modified with a 5′ guanosine monophosphorothioate (GMPS) functional unit and the target for which a ligand is obtained is N-bromoacetylated-bradykinin (BrBK). This example describes the selection and analysis of a 5′ guanosine monophosphorothioate-substituted RNA (GMPS-RNA) which specifically recognizes N-bromoacetylated-bradykinin (BrBK) and accelerates the formation of a thioether bond between the RNA and the BrBK peptide. Previous work in this area showed that it was difficult to obtain ligands to bradykinin both in free solution and conjugated to a support matrix. As will be described below, RNA showing a 6700-fold increase in kcat / Km and a 100-fold increase in binding affinity for N-bromoacetyl-bradykinin relative to the starting pool was identified. This RNA binds its substrate with high specificity, requiring both the amino- and carboxy-ter...

example 2

Splint SELEX to Identify Elastase Inhibitors

[0078] Highly potent and specific inhibitors of human neutrophil elastase were produced by an approach that incorporates the technologies of medicinal and combinatorial chemistry. A small-molecule covalent inhibitor of elastase (the valyl phosphonate functional unit) was coupled to a randomized pool of RNA, and this assembly was iteratively selected for sequences that promote a covalent reaction with the elastase target active site. The winning sequences increase both the binding affinity and reactivity over that of the small molecule functional unit alone; the overall increase in the second-order rate of inactivation was ˜104-fold. The rate of cross-reaction with another serine protease, cathepsin G, was reduced>100-fold. These compounds inhibit elastase expressed from induced human neutrophils, and prevent injury in an isolated rat lung model of ARDS. This strategy is generally useful for increasing the potency and specificity of small...

example 3

Nucleic Acid Ligands That Bind to HIV-1 Rev Protein

[0105] A target protein chosen to illustrate photo-SELEX process described in copending PCT / US94 / 10562 (WO 95 / 08003), filed Sep. 16, 1994, which is herein incorporated by reference was theRev protein from HIV-1. The example provided herein describes that ligands were identified which bound covalently to the Rev protein both with and without irradiation.

[0106] Rev's activity in vivo is derived from its association with the Rev-responsive element (RRE), a highly structured region in the HIV-1 viral RNA. Previous RNA SELEX experiments of Rev have allowed the isolation of very high affinity RNA ligands. The highest affinity ligand, known as “Rev 6a,” (SEQ ID NO: 103) has a Kd of approximately 1 nM. The sequence of Rev 6a is

(SEQ ID NO: 103)GGGUGCAUUGAGAAACACGUUUGUGGACUCUGUAUCU.

The secondary structure of 6a, and its interaction with Rev, have been well characterized.

[0107] The construction of the nucleic acid test mixture for photo...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
pHaaaaaaaaaa
pHaaaaaaaaaa
pHaaaaaaaaaa
Login to view more

Abstract

This application provides methods for identifying nucleic acid ligands capable of covalently interacting with targets of interest. The nucleic acids can be associated with various functional units. The method also allows for the identification of nucleic acids that have facilitating activities as measured by their ability to facilitate formation of a covalent bond between the nucleic acid, including its associated functional unit, and its target.

Description

RELATED APPLICATIONS [0001] This application is a continuation of U.S. patent application Ser. No. 09 / 974,330, filed Oct. 9, 2001, which is a continuation of U.S. patent application Ser. No. 09 / 412,017, filed Oct. 4, 1999, now U.S. Pat. No. 6,300,074, which is a continuation of U.S. patent application Ser. No. 08 / 460,888, filed Jun. 5, 1995, now U.S. Pat. No. 5,962,219, which is a continuation of U.S. patent application Ser. No. 08 / 400,440, filed Mar. 8, 1995, now U.S. Pat. No. 5,705,337, each entitled “Systematic Evolution of Ligands by Exponential Enrichment: Chemi-Selex,” which is a continuation-in-part of U.S. patent application Ser. No. 07 / 714,131, filed Jun. 10, 1991, entitled “Nucleic Acid Ligands,” now U.S. Pat. No. 5,475,096, which was filed as a continuation-in-part of U.S. patent application Ser. No. 07 / 536,428, filed Jun. 11, 1990, entitled “Systematic Evolution of Ligands by Exponential Enrichment,” now abandoned. This application is also a continuation-in-part of U.S. ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12P19/34C07H21/04C07B61/00C07H19/06C07H19/10C07H21/00C07K14/00C12N9/12C12N15/10C12N15/115C12Q1/37C12Q1/70F02B75/02G01N33/531G01N33/532G01N33/535G01N33/569G01N33/68G01N33/76
CPCA61K47/48076A61K47/48092B82Y5/00C07H19/06C07H19/10C07H21/00C07K14/001C12N9/1276C12N15/1048C12N15/115C12N2310/13C12N2310/322C12N2310/53C12P19/34C12Q1/37C12Q1/6804C12Q1/6811C12Q1/6876C12Q1/70C40B40/00F02B2075/027G01N33/531G01N33/532G01N33/535G01N33/56988G01N33/68G01N33/76G01N2333/163G01N2333/575G01N2333/62G01N2333/8125G01N2333/96433G01N2333/96455G01N2333/966G01N2333/9726G01N2333/974G01N2333/976C12Q2541/101C12Q2525/101C12Q2525/179C12Q2525/155C12Q2521/101A61K47/547A61K47/549
Inventor GOLD, LARRYEATON, BRUCESMITH, DREWWECKER, MATTHEWJENSEN, KIRK
Owner GILEAD SCI INC
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap