Higher order structure and binding of peptide nucleic acids

Details Higher order structure and binding of peptide nucleic acids Higher order structure and binding of peptide nucleic acids

1999-11-16

2004-08-03

Marschel, Ardin H. (Department: 1631)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

C435S006120, C514S002600, C514S04400A, C530S333000, C530S350000

Reexamination Certificate

active

06770738

ABSTRACT:

FIELD OF THE INVENTION
This invention is directed to compounds that form triple-stranded structures with single-stranded and double-stranded nucleic acids. It is further directed to the use of such compounds to cause strand displacement in double-stranded nucleic acids. The invention further is directed to processes for modifying double-stranded nucleic acid utilizing such strand displacement. Such processes for modifying double-stranded nucleic acids include cleavage of the nucleic acid strand or strands. In particular, such cleavage includes sequence specific cleavage of double-stranded nucleic acids using a nuclease which normally is nonsequence specific. Such process also include transcription inhibition or arrest as well as transcription initiation. The processes of the invention are effected, in particular, with compounds that include naturally-occurring nucleobases or other nucleobase-binding moieties covalently bound to a polyamide backbone.
BACKGROUND OF THE INVENTION
The function of a gene starts by transcription of its information to a messenger RNA (mRNA) which, by interaction with the ribosomal complex, directs the synthesis of a protein coded for by the mRNA sequence. The synthetic process is known as translation. Translation requires the presence of various co-factors and building blocks, the amino acids, and their transfer RNAs (tRNA), all of which are present in normal cells.
Transcription initiation requires specific recognition of a promoter DNA sequence by the RNA-synthesizing enzyme, RNA polymerase. In many cases in prokaryotic cells, and probably in all cases in eukaryotic cells, this recognition is preceded by sequence-specific binding of a protein transcription factor to the promoter. Other proteins which bind to the promoter, but whose binding prohibits action of RNA polymerase, are known as repressors. Thus, gene activation typically is regulated positively by transcription factors and negatively by repressors.
Most conventional drugs function by interaction with and modulation of one or more targeted endogenous proteins, e.g., enzymes. Typical daily doses of drugs are from 10
−5
-10
−1
millimoles per kilogram of body weight or 10
−3
-10 millimoles for a 100 kilogram person. If this modulation instead could be effected by interaction with and inactivation of mRNA, a dramatic reduction in the necessary amount of drug necessary likely could be achieved, along with a corresponding reduction in side effects. Further reductions could be effected if such interaction could be rendered site-specific. Given that a functioning gene continually produces mRNA, it would thus be even more advantageous if gene transcription could be arrested in its entirety.
Synthetic reagents that bind sequence selectively to single-stranded and especially to double-stranded nucleic acids are of great interest in molecular biology and medicinal/chemistry, since such reagents may provide the tools for developing gene-targeted drugs and other sequence-specific gene modulators. Until now oligonucleotides and their close analogues have presented the best candidates for such reagents.
Oligodeoxyribonucleotides as long as 100 base pairs (bp) are routinely synthesized by solid phase methods using commercially available, fully automatic synthesis machines. Oligoribonucleotides, however, are much less stable than oligodeoxyribonucleotides, a fact which has contributed to the more prevalent use of oligodeoxyribonucleotides in medical and biological research directed to, for example, gene therapy and the regulation of transcription and translation. Synthetic oligodeoxynucleotides are being investigated for used as antisense probes to block and eventually breakdown mRNA.
It also may be possible to modulate the genome of an animal by, for example, triple helix formation using oligonucleotides or other DNA recognizing agents. However, there are a number of drawbacks associated with oligonucleotide triple helix formation. For example, triple helix formation generally has only been obtained using homopurine sequences and requires unphysiologically high ionic strength and low pH. Whether used as antisense reagents or a triplexing structures, unmodified oligonucleotides are unpractical because they have short in vivo half-lives. To circumvent this, oligonucleotide analogues have been used.
These areas for concern have resulted in an extensive search for improvements and alternatives. For example, the problems arising in connection with double-stranded DNA (dsDNA) recognition through triple helix formation have been diminished by a clever “switch back” chemical linking whereby a sequence of polypurine on one strand is recognized, and by “switching back”, a homopurine sequence on the other strand can be recognized. See, e.g., McCurdy, Moulds, and Froehler,
Nucleosides,
in press. Also, helix formation has been obtained by using artificial bases, thereby improving binding conditions with regard to ionic strength and pH.
In order to improve half-life as well as membrane penetration, a large number of variations in polynucleotide backbones has been undertaken. These variations include the use of methylphosphonates, monothiophosphates, dithiophosphates, phosphoramidates, phosphate esters, bridged phosphoramidates, bridged phosphorothioates, bridged methylenephosphonates, dephospho internucleotide analogs with siloxane bridges, carbonate bridges, carboxymethyl ester bridges, acetamide bridges, carbamate bridges, thioether, sulfoxy, sulfono bridges, various “plastic” DNAs, &agr;-anomeric bridges, and borane derivatives. The great majority of these backbone modifications have decreased the stability of hybrids formed between a modified oligonucleotide and its complementary native oligonucleotide, as assayed by measuring T
m
values. Consequently, it is generally believed in the art that backbone modifications destabilize such hybrids, i.e., result in lower T
m
values, and should be kept to a minimum.
The discovery of sequence specific endonucleases (restriction enzymes) was an essential step in the development of biotechnology, enabling DNA to be cut at precisely specified locations containing specific base sequences. However, although the range of restriction enzymes now known is extensive, there is still a need to obtain greater flexibility in the ability to recognize particular sequences in double-stranded nucleic acids and to cleave the nucleic acid specifically at or about the recognized sequence.
Most restriction enzymes recognize quartet or sextet DNA sequences and only a very few require octets for recognition. However, restriction enzymes have been identified and isolated only for a small subset of all possible sequences within these constraints. A need exists, especially in connection with the study of large genomic DNA molecules, in general, and with the human genome project, in particular, to recognize and specifically cleave DNA molecules at more rarely occurring sites, e.g., sites defined by about fifteen base pairs.
Efforts have therefore been made to create artificial “restriction enzymes”or to modify the procedures for using existing restriction enzymes for this purpose. Methods investigated include the development of oligonucleotides capable of binding sequence specifically via triple helix formation to double-stranded DNA tagged with chemical groups (e.g., photochemical groups) able to cleave DNA or with non-specific DNA cleaving enzymes and other such modifications consistent with an “Achilles heel” general strategy. Such methods are described by: Francois, J. C., et al. PNAS 86,9702-9706 (1989); Perrouault, L., et al. Nature 344,358-360 (1990); Strobel, S. A. & Dervan P. B. Science 249,73-75 (1990); Pei, D., Corey D. R. & Schultz P. G. PNAS 87,9858 (1990); Beal, P. A. & Dervan P. B., Science 251,1360 (1991); Hanvey, J. C., Shimizu M. & Wells R. D. NAR 18,157-161 (1990); Koob, H. & Szybalski W. Science 250,271 (1990); Strobel, S. A. & Dervan P. B. Nature #50,172 (1991); and Ferrin, L. J. & Camerini-Otero R. D. Science 254,1494-1497 (1991).
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