Binding proteins for recognition of DNA

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...

AI Technical Summary

Benefits of technology

[0017]Preferably the zinc finger binding motif is present within the context of other amino acids (which may be present in zinc finger proteins), so as to form a zinc finger (which includes an antiparallel β-sheet). Further, the zinc finger is preferably displayed as part of a zinc finger polypeptide, which polypeptide comprises a plurality of zinc fingers joined by an intervening linker peptide. Typically the library of sequences is such that the zinc finger polypeptide will comprise two or more zinc fingers of defined amino acid sequence (generally the wild type sequence) and one zinc finger having a zinc finger binding motif randomised in the manner defined above. It is preferred that the randomised finger of the polypeptide is positioned between the two or more fingers having defined sequence. The defined fingers will establish the “phase” of binding of the polypeptide to DNA, which helps to increase the binding specificity of the randomised finger.

[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 9 bases long then three zinc finger binding motifs can be included in the polypeptide. If the known sequence is 27 bases long then, in theory, up to nine binding motifs could be included in the polypeptide. The longer the target DNA sequence, the lower the probability of its occurrence in any given portion of DNA.

[0032]Moreover, those motifs selected for inclusion in the polypeptide could be artificially modified (e.g. by directed mutagenesis) in order to optimise further their binding characteristics. Alternatively (or additionally) the length and amino acid sequence of the linker peptide joining adjacent zinc binding fingers could be varied, as outlined above. This may have the effect of altering the position of the zinc finger binding motif relative to the DNA sequence of interest, and thereby exert a further influence on binding characteristics.

[0043]Binding of the zinc finger polypeptide to the target sequence may result in increased or reduced expression of the gene of interest depending, for example, on the nature of the target sequence (e.g. structural or regulatory) to which the polypeptide binds.

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 misconstrued.

Six triplets were used in selections but did not return fingers with any sequence biases; and when the three triplets of the Zif268 binding site were individually used as controls, the vast majority of selected fingers did not resemble the sequences of the wild-type Zif268 fingers and, though capable of tight binding to their target sites in vitro, were usually not able to discriminate strongly against different triplets.

A recognition code, to aid design of new finger specificities, has been worked towards although it has been suggested that specificity may be difficult to predict.

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