Compositions, methods and kits for identifying naturally...

Details Compositions, methods and kits for identifying naturally... Compositions, methods and kits for identifying naturally...

1999-02-22

2001-05-29

Jones, W. Gary (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S091520, C435S091510, C536S022100

Reexamination Certificate

active

06238867

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to methods, compositions, kits and apparatus to identify ribonucleic acid (RNA) molecules having a consensus sequence exhibiting affinity for RNA binding proteins. And, the present invention is directed to compositions, methods, kits and apparatus to identify proteins which exhibit affinity to consensus sequences of RNA molecules which have affinity for RNA-binding proteins. Embodiments of the present invention have utility in the identification of RNA-binding proteins and/or RNA molecules having affinity for consensus sequences of RNA-binding proteins. Embodiments of the present invention have utility in the determination of the presence, absence or concentration of such RNA molecules and or such proteins in specimens and samples.
BACKGROUND OF THE INVENTION
In living cells, RNA is normally associated with proteins to form various nucleoprotein complexes (RNPs). These RNAs are sometimes referred to as splicosomal RNA; ribosomal RNA (rRNA); RNA-associated enzymes, such as RNAses, RNA ligases and telomerase; messenger RNA (mRNA); transfer RNA (tRNA); heterogenous nuclear RNA (hnRNA); and small nuclear RNA (snRNA). The RNPs corresponding to such RNAs are commonly referred to with reference to similar or identical prefix designations, as in heterogenous nuclear protein complex or, simply, hnRNP. The protein component of the RNP is commonly referred to with reference to such prefix and the complex, as in heterogenous nuclear protein complex protein or, simply, hnRNP protein.
A significant portion of RNA-binding proteins mediate the post-transcriptional regulation of gene expression. Heterogenous nuclear RNAs (hnRNA) are the primary transcripts of protein coding genes. hnRNAs are processed in the nuclei of eukariotic cells and, at least a portion of such hnRNAs, become mRNAs. From the time hnRNAs emerge from the transcriptional complex, and throughout the time they are in the nucleus, they are associated with proteins termed as hnRNP proteins. Members of this family of proteins are required for multiple steps during mRNA metabolism, including pre-mRNA processing and mRNA localization, translation and stability. The majority of proteins associated with RNAs appear to be associated with hnRNAs and messenger RNAs (mRNAs) in hnRNP and mRNP complexes. However, there are numerous, less abundant proteins which are associated with other groups of RNAs (Dreyfuss et al., 1993).
Clearly, hnRNPs and other RNP complexes have great importance for cell function. And, the presence or absence of such RNP complexes, or abnormalities in such RNP complexes may have substantial implications in disease. By way of example, without limitation, the presence or absence or abnormalities in RNP complexes may be an indication of an inherited disease, cancer, prion disease, and age related disease.
Viral RNPs and their RNA and protein components define much of the disease processes associated with the virus pathogens (e.g.HIV, Dengue virus, etc.,). By way of example two viral enzymes, Tat and rev proteins, are essential in a life cycle of HIV and form an RNP. RNPs and their RNA and protein components, for both RNA and DNA viruses, have great value as diagnostic tools.
Bacterial and viral RNP complexes are attractive diagnostic targets. The RNP complexes of pathogens, and associated RNA molecules and protein components, may present much more numerous copies in a sample compared to genomic or ribosomal targets.
A need exists for analytical methods, compositions, kits and apparatus to identify and characterize new RNP complexes. Such analytical methods, compositions, kits and apparatus have utility in the diagnosis of various cancers, infectious and inherited diseases.
SUMMARY OF INVENTION
Embodiments of the present invention features methods, compositions, kits, and apparatus for identification and characterization of the RNA sequences having specific affinity to amino acid consensus sequences of RNA-binding proteins. And, embodiments of the present invention feature such means for the identification and characterization of proteins having amino acid consensus sequences having specific affinity to RNA.
As used herein, the term “consensus sequence” means RNA-binding motifs that recognize single stranded RNA secondary structural elements such as hairpin loops, bulge loops, internal loops or single-stranded regions. Most of RNP proteins have a modular structure with one or more RNA-binding domains (RBD) and one another domain that mediates interaction with another protein. The hallmarks of the RNP motif are consensus sequences located about 30 amino acids apart in RBD, composed of from hundred to several hundreds of amino acids. Most of amino acids that participate in RNA binding are located in beta-sheet surface and these structural elements of RBD appear to provide an exposed platform to which RNA binds. The RNA, when bound, remains exposed (as opposed to buried in a fold or a pocket) and accessible to other RNA processing factors. Many RNP proteins contain multiple RBDs, and can bind to more than one RNA molecule simultaneously (Kiledjian et al., 1994).
As used herein, the term “affinity” means exhibiting an attraction or capable of binding. A specific affinity is an attraction which is directed to a particular feature or sequence of a molecule.
One embodiment of the present invention is a composition. The composition comprises a first ribonucleic acid (RNA) molecule and a second RNA molecule. The first RNA molecule is capable of binding to a RNA-binding protein and has the following formula:
5′-A-B-C-3′.
As used above, the letter “A” represents a section of the RNA molecule having 10-100,000 nucleotides, which section can be received by an RNA replicase and with another RNA sequence, F, being replicated. The letter “B” denotes a section of the RNA molecule having approximately 10 to 3,000 and more preferred 15 to 1,000, and more preferred 30 to 100 nucleotides in a first sequence having an affinity to at least one amino acid consensus sequence of RNA-binding protein, which section is capable of binding to such protein molecule. The letter “C” denotes a section of the RNA molecule having approximately 1 to 20 nucleotides which section is capable of being ligated to another RNA sequence, “D”. The second RNA molecule is capable of binding to a RNA-binding protein and has the following formula:
5′-D-E-F-3′.
As used above, the letter “D” is a section of the RNA molecule having approximately 1 to 20 nucleotides, which section is capable of being ligated to another RNA sequence, “C”. The letter “E” denotes a section of the RNA molecule having approximately 10 to 3000, and more preferred 15 to 1,000, and more preferred 30 to 100, nucleotides in a second sequence having an affinity to another amino acid consensus sequence of the same RNA-binding protein to which the section B exhibits affinity. Section E is capable of binding to such RNA-binding protein. The letter “F” denotes a section of the RNA molecule having 10-100,000 nucleotides which section is capable of being received by an RNA replicase and with another sequence, “A”, being replicated. The first and the second RNA molecules are differ in their composition, such as they have two different parts of RNA replicase template. Neither of these parts can serve separately as an amplifiable template for RNA replicase.The first and the second RNA molecules capable of forming a third, hybrid RNA molecule having the following formula:
5′-A-B-C-D-E-F-3′.
The third RNA molecule is formed by ligating the C and D sections, as the E and the B sections are bound to the two consensus sequences of the same RNA-binding protein. The third RNA molecule is capable of being received by an RNA replicase and being replicated by such enzyme.
The sequences represented by the letters A, B, C, D, E, and F may further comprise sequences and nucleotides which are artifacts of cloning, synthesis or naturally occurring nucleotides.
Preferably, the sequences represented by the letters “A” and “F” are

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