Methods for modulating telomerase activity

Abstract

The present invention relates to isolated or purified molecule(s) capable of binding to one or more of telomeric, G-quadruplex, or G-quartet nucleic acid(s).

Description

[0001] The invention relates to DNA binding molecules. In particular the invention relates to molecules which bind to G-quadruplex or telomeric DNA.BACKGROUND TO THE INVENTION[0002] There is considerable interest in molecules that bind to telomeric DNA sequences and G-quadruplexes. Such molecules will be useful to test hypotheses of telomere length regulation, and may have therapeutic potential.[0003] Several naturally occurring proteins with affinity for G-quadruplexes have been described in the prior art (reviewed in Wellinger. R. J., & Sen, D. (1997) European Journal of Cancer 33, 735-749), although none have so far proved to be good candidates for use as diagnostic probes or therapeutic tools.[0004] Prior art quadruplex DNA binding molecules, such as a recently reported DNA-binding autoantibody (Brown, B. A., Li, Y. Q., Brown, J. C., Hardin, C. C., Roberts, J. F., Pelsue, S. C., & Shultz., L. D. (1998) Biochemistry 37, 16325-16337), have only moderate binding affinities and disc...

AI Technical Summary

Benefits of technology

[0063] Promoters suitable for use with prokaryotic hosts include, for example, the .beta.-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (Trp) promoter system and hybrid promoters such as the tac promoter. Their nucleotide sequences have been published, thereby enabling the skilled worker operably to ligate them to DNA encoding nucleic acid binding protein, using linkers or adapters to supply any required restriction sites. Promoters for use in bacterial systems will also generally contain a Shine-Delgarno sequence operably linked to the DNA encoding the nucleic acid binding protein.

[0089] The the present invention facilitates ELISA-based detection of telomerase activity. This detection system is rapid, easily automated with liquid handling robotics and avoids the need to use radioactivity. This contrasts with prior art telomerase assays such as the commercially-available `TRAP` assay.

[0093] The present invention facilitates the construction of ELISA-based diagnostic kits for the detection of telomerase activity. These assays are rapid, easily automated with liquid handling robotics and avoid the need to use radioactivity, in contrast to prior art technologies such as the `TRAP` assay.

[0097] Zinc finger protein molecules according to the invention may be selected from a phage display library to bind G-quadruplex DNA structures of single stranded human telomeric sequences with selectivity and high affinity. Advantageously, these zinc fingers have no detectable affinity for a duplex DNA made up of the Htelo sequence and its complementary strand. These molecules represent a new class of DNA-binding zinc lingers and have utility for both study and exploration of the molecules themselves, and of therapeutics and assays, in addition to their utility as in vitro or in vivo molecular probes to explore possible mechanisms of inhibition and regulation of telomerase-mediated telomere extension. The widespread conservation of G-quadruplex-forming sequences at chromosome ends means that the molecules according to the invention will find utility in a wide range of biological systems.

[0121] The present invention has advantages over existing technology which include but are not limited to increased speed and sensitivity when using amplifying ELISA signal, removal of the need for running electrophoretic gels, and alleviation of the need to use radioactive labelling. Furthermore, the systems of the present invention can be advantageously automated using liquid-handling robotics, resulting in high efficiency and labour-saving.

Problems solved by technology

For example, a vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells even though it is not capable of replicating independently of the host cell chromosome.

However, the recovery of genomic DNA encoding the nucleic acid binding protein is more complex than that of exogenously replicated vector because restriction enzyme digestion is required to excise nucleic acid binding protein DNA.

), although there are problems associated with their use as diagnostic or therapeutic probes.

Furthermore, non-conventional DNA structures include non-double helical DNA conformations.

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