Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-10-25
2004-06-01
Zitomer, Stephanie W. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C436S501000, C530S300000, C530S350000, C530S387100, C536S023100, C536S024300
Reexamination Certificate
active
06743586
ABSTRACT:
FIELD OF THE INVENTION
This invention pertains to a method for selective substitution of highly fluorescent nucleotide base analogs within a sequence of nucleic acid drug targets. The substitution is particularly selected such that the base can be used as a probe to monitor and screen for interactions of ligands with a nucleic acid target.
BACKGROUND
The development of specific inhibitors of protein-RNA interactions, that are unique and required for retroviral viability, is of significant interest to pharmaceutical industries since these complexes provide new and potentially powerful targets to inhibit retroviral infection.
As described in U.S. Pat. No. 6,153,382, herein incorporated by reference, the HIV genome is tightly compressed. At least 30 different RNA transcripts are produced by splicing using the six splice acceptors and two splice donor sequences. The structural proteins encoded by HIV are chemically similar to those of the C-type retroviruses and, like them, are encoded as polyproteins by the gag (group antigen), pol (polymerase) and env (envelope) genes. Cleavage of the polyproteins by the viral protease or cellular enzymes generates eight functional virion proteins. In addition to these structural genes, HIV-1 also carries genes for three regulatory proteins, rev (regulator factor); and two proteins involved in virus maturation, vif (virion infectivity factor) and vpu (viral protein U). The vpr (viral protein R) gene encodes a low copy number virion component. In the closely related viruses HIV-2 and simian immunodeficiency virus (SIV), vpr is replaced by vpx (viral protein X), a unique virion protein.
Transcription of the HIV genome during virus replication shows distinct kinetic phases. The initial products of HIV gene expression are short, multiply spliced mRNAs approximately 1.8 to 2.0 kb in length, which encode the trans-acting regulatory proteins including rev. As infection by the virus develops, and the levels of the rev protein rise in the infected cells, mRNA production shifts progressively towards synthesis of a family of singly-spliced 4.3 kb mRNAs encoding env and other HIV gene products such as vif and vpr. Finally, late in the infection process, production switches to full-length, unspliced, transcripts that act both as the virion genomic RNA and the mRNA for the gag-pol polyprotein.
To achieve this control of gene expression, HIV relies on the interaction of cellular and virus-encoded trans-acting factors with cis-acting viral regulatory sequences. Initiation of transcription relies largely on the presence of binding sites for cellular transcription factors in the viral long terminal repeat (LTR). In contrast, the virally encoded regulatory proteins tat and rev exert their activity via cis-acting sequences encoded within HIV messenger RNAs. After a systematic search, a cis-acting sequence required for rev activity, was mapped to a complicated RNA stem-loop structure located within the env reading frame. This sequence has been named the rev-responsive element (RRE) and has been localized to a 234-nucleotide long sequence within the env gene. Similar regulatory proteins and target sequences are used by HIV-2 and SIV. The human T-cell leukemia (HTLV-1) virus rex gene product appears to function analogously to rev, and can functionally substitute for rev to promote viral gene expression.
The distinct kinetic phases of HIV transcription are now believed to reflect the intracellular levels of the regulatory proteins tat and rev. Initially, binding of host transcription factors to the LTR induced basal level transcription of the early mRNAs including tat. As tat levels rise, increased transcription from the LTR is stimulated by the trans-activation mechanism. This leads to further increases in tat levels, and also stimulates production of rev. Production of the viral structural proteins begins once rev levels have risen to sufficiently high levels to promote export of messenger RNAs carrying the RRE sequence. The HIV growth cycle may also include a latent stage where viral gene expression is silent because transcription from the viral LTR produces insufficient amounts of regulatory proteins to initiate the lytic growth cycle.
Rev recognition of the RRE RNA element, like tat recognition of HIV-1 trans activation region (TAR) RNA, is due to direct binding. Binding is tight (K
D
=1-3 nM) and highly specific for the RRE. However, the binding behavior of rev to RRE is much more complex than the binding of tat to TAR RNA. As the concentration of rev increases, progressively larger complexes with RRE RNA are formed, whereas tat only forms one-to-one complexes with TAR RNA.
The simplest explanation for the RNA binding behavior of rev is that the protein binds initially to a high affinity site and that subsequently additional rev molecules occupy lower affinity sites.
Many methods for nucleic acid-protein binding assays, to measure interactions of, for example, RRE with rev, have been developed, such as gel electrophoresis, filter binding and chromatography. However, these procedures lack sufficient speed, sensitivity and accuracy to be useful as high-throughput binding assays. Additionally, fluorescence methods, such as fluorescence resonance energy transfer (FRET), provide many advantages, however do not detect both direct and indirect interactions of rev peptide or ligand binding that cause subtle conformational changes in the nucleic acid structure of RRE.
SUMMARY OF THE INVENTION
The present invention pertains to a generally useful approach for detecting and quantifying the interaction of nucleic acids with macromolecules and/or small molecule ligands that can be adapted into high-throughput screens (HTS). A fluorescence-based binding assay, i.e. fluorescence emission perturbation (FREP) has been developed that provides such a method for screening and optimizing inhibitors of nucleic-acid protein interactions that are potential and so far unexploited targets for anti-viral and anti-cancer drugs. The FREP method described and claimed herein, provides a general, rapid and sensitive fluorescence assay that allows the direct detection and quantification of the interaction of ligand with nucleic acid targets. Using the FREP assay, multiple binding classes of small molecules to nucleic acid targets can be directly detected, as well as specific inhibition of macromolecular interactions with nucleic acid targets by small molecule antagonists. The ability to detect both macromolecular (mostly protein) and small molecule binding using the same fluorescently labeled nucleic acid constructs allows the application of dual screening; where analysis for both direct binding of small molecules, as well as the potential competitive inhibition of the nucleic acid-protein interaction by these molecules may be assayed simultaneously.
In particular, the FREP binding assay, in one form, includes an RRE oligonucleotide labeled with the fluorescence adenosine analog, 2-aminopurine (2-AP) in specific nucleotide. Modified RRE oligonucleotides show measurable changes in fluorescence emission that can be directly correlated with rev and small molecule binding to RRE; thereby providing a general method for monitoring these binding interactions. Rev monomers (or small peptides derived from rev) act by first binding to a high affinity site within the response element (RRE), a 34-nucleotide stem-loop structure (stem-loop IIB) found in nuclear HIV-1 mRNA transcripts (FIG.
1
).
The FREP method is applicable in the context of many nucleic acid systems. Specifically, however, the FREP method is particularly advantageous for use in measuring interactions with (1) the above-described HIV-1 RRE-rev system; (2) HIV-1 dimerization initiation sequences (DIS); and (3) topoisomerase DNA systems.
Previous studies have shown the 2-AP can be used as a valuable probe of the structure and dynamics of specific sites in DNA, as a monitor for enzyme-DNA interaction, and to study Mg
2+
dependent conformational changes in certain ribozymes. The fluorescence of 2-AP is usually highly quenched when i
Lacourciere Karen
Marino John P.
Stivers James T.
The United States of America as represented by the Secretary of
Zitomer Stephanie W.
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