Binding proteins for recognition of DNA

Inactive Publication Date: 2006-08-08
GENDAQ +1
View PDF63 Cites 29 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0031]It has now been shown possible by the present inventors (below) to design a truly modular zinc binding polypeptide, wherein the zinc binding motif of each zinc binding finger is selected on the basis of its affinity for a particular triplet. Accordingly, it should be well within the capability of one of normal skill in the art to design a zinc finger polypeptide capable of binding to any desired target DNA sequence simply by considering that sequence of triplets present in the target DNA and combining in the appropriate order zinc fingers comprising zinc finger binding motifs having the necessary binding characteristics to bind thereto. The greater the length of known sequence of the target DNA, the greater the number of zinc finger binding motifs that can be included in the zinc finger polypeptide. For example, if the known sequence is only

Problems solved by technology

They state “there is no prospect of achieving a zinc finger recognition code”.
They state that the design of Zf proteins with predictable specificities and affinities “may not always be straightforward”.
However in this approach the altered positions on the α-helix are prejudged, making it possible to overlook the role of positions which are not currently considered important; and secondly, owing to the importance of context, concomitant alterations are sometimes required to affect specificity (Desjarlais & Berg 1992b), so that a significant correlation between an amino acid and base may be mi

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
  • Binding proteins for recognition of DNA
  • Binding proteins for recognition of DNA
  • Binding proteins for recognition of DNA

Examples

Experimental program
Comparison scheme
Effect test

Example

EXAMPLE 1

[0077]In this example the inventors have used a screening technique to study sequence-specific DNA recognition by zinc finger binding motifs. The example describes how a library of zinc finger binding motifs displayed on the surface of bacteriophage enables selection of fingers capable of binding to given DNA triplets. The amino acid sequences of selected fingers which bind the same triplet were compared to examine how sequence-specific DNA recognition occurs. The results can be rationalised in terms of coded interactions between zinc fingers and DNA, involving base contacts from a few α-helical positions.

[0078]An alternative to the rational but biased design of proteins with new specificities, is the isolation of desirable mutants from a large pool. A powerful method of selecting such proteins is the cloning of peptides (Smith 1985 Science 228, 1315-1317), or protein domains (McCafferty et al., 1990 Nature (London) 348, 552-554; Bass et al., 1990 Proteins 8, 309-314), as f...

Example

EXAMPLE 2

[0105]This example describes a new technique to deal efficiently with the selection of a DNA binding site for a given zinc finger (essentially the converse of example 1). This is desirable as a safeguard against spurious selections based on the screening of display libraries. This may be done by screening against libraries of DNA triplet binding sites randomised in two positions but having one base fixed in the third position. The technique is applied here to determine the specificity of fingers previously selected by phage display. The inventors found that some of these fingers are able to specify a unique base in each position of the cognate triplet. This is further illustrated by examples of fingers which can discriminate between closely related triplets as measured by their respective equilibrium dissociation constants. Comparing the amino acid sequences of fingers which specify a particular base in a triplet, we infer that in most instances, sequence specific binding o...

Example

EXAMPLE 3

[0133]From the evidence presented in the preceding examples, the inventors propose that specific DNA-binding proteins comprising zinc fingers can be “made to measure”. To demonstrate their potential the inventors have created a three finger polypeptide able to bind site-specifically to a unique 9 bp region of a BCR-ABL fusion oncogene and to discriminate it from the parent genomic sequences (Kurzrock et al., 1988 N. Engl. J. Med. 319, 990 -988). Using transformed cells in culture as a model, it is shown that binding to the target oncogene in chromosomal DNA is possible, resulting in blockage of transcription. Consequently, murine cells made growth factor-independent by the action of the oncogene (Daley et al., 1988 Proc. Natl. Acad. Sci. U.S.A. 85, 9312-9316) are found to revert to factor dependence on transient transfection with a vector expressing the designed zinc finger polypeptide.

[0134]DNA-binding proteins designed to recognise specific DNA sequences could be incorpor...

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
Affinityaaaaaaaaaa
Login to view more

Abstract

Disclosed are libraries of DNA sequences encoding zinc finger binding motifs for display on a particle, together with methods of designing zinc finger binding polypeptides for binding to a particular target sequence and, inter alia, use of designed zinc finger polypeptides for various in vitro or in vivo applications.

Description

[0001]This application is the national phase of international application PCT / GB95 / 01949, filed Aug. 17, 1995 which designated the U.S.CROSS-REFERENCE TO RELATED APPLICATIONS [0002]This application is a Reissue of U.S. Pat. No. 6,007,988, which issued on Dec. 28, 1999 from U.S. Ser. No. 08 / 793,408, filed on Jun. 2, 1997, which was a National Phase filing pursuant to 35 U.S.C. §371 of PCT / GB95 / 01949 filed on Aug. 17, 1995. PCT / GB95 / 01949 in turn claims priority to GB 9514698.1(filed Jul. 18, 1995), GB 9422534.9(filed Nov. 8, 1994) and GB 9416880.4(filed Aug. 20, 1994). The foregoing are all incorporated by reference in their entireties herein. More than one Reissue of U.S. Pat. No. 6,007,988 has been filed, including the present application, a continuation of the present application having U.S. Ser. No. 10 / 309,578(filed Dec. 3, 2002), a divisional of the present application having U.S. Ser. No. 10 / 397,930(filed Mar. 25, 2003) and a divisional of the present application having U.S. Se...

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
IPC IPC(8): C12Q1/68C12N15/62C12N15/63C12N5/02C12P21/02C12N15/09A61K38/00A61K48/00A61P35/00C07K14/47C12N15/10C12N15/12C40B40/02
CPCC07K14/4702C07K14/4705C07K2319/00C07K2319/30C07K2319/81C12N15/1037C12Q1/68C40B40/02C07K19/00C07K2319/09A61P35/00
Inventor CHOO, YENKLUG, AARONSANCHEZ-GARCIA, ISIDRO
Owner GENDAQ
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