Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-11-09
2001-03-27
Houtteman, Scott W. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100
Reexamination Certificate
active
06207388
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to methods, compositions, kits and apparatus to identify and detect the presence or absence of target analytes. The embodiments of the present invention have utility in medical diagnosis and analysis of various chemical compounds in specimens and samples, as well as the design of test kits and apparatus for implementing such methods.
BACKGROUND OF THE INVENTION
Molecular biology advances in the last decade gave great promise for the introduction of new, sensitive technologies to identify various analytes in test specimens, including the ability to diagnose cancer, infectious agents and inherited diseases. Clinical molecular diagnostics depend almost exclusively on restriction enzyme analyses and nucleic acid hybridization (Southern and Northern blots) (Meselson and Yuan, 1968, Southern, 1975). Clinical tests based on molecular biology technology are more specific than conventional immunoassay procedures and can discriminate between genetic determinants of two closely related organisms. With their high specificity, nucleic acid procedures are very important tools of molecular pathology. However, nucleic acid procedures have limitations, the most important of which are the procedures consume time, they are labor intensive, and have low sensitivity (Nakamura 1993).
In any sample, the number of protein molecules of one kind is usually several times higher than the corresponding mRNA, and several hundred times higher than the number of genes encoding them. Using antigen-specific antibodies is a routine procedure in the modem diagnostic industry, although antibody development and purification usually require laborious work. The specificity of tests based on monoclonal antibodies depends on the capacity of antibodies to differentiate between antigens, and might approach the specificity of tests based on nucleic acid hybridization. The sensitivity of these tests, however, is routinely significantly lower than tests based on nucleic acid hybridization, even though the number of protein target molecules in each cell is relatively higher than the nucleic acid molecules corresponding to them. It is desirable to use proteins as the targets in diagnostic tests because of their abundance.
Thus, a need exists for improved diagnostic and analytical methods to detect the presence or absence of target molecules. A need also exists to detect non-nucleic acid analytes with nucleic acid chemistry. And, there is a need to detect protein targets with nucleic acid chemistry coupled to a amplification system.
SUMMARY OF INVENTION
The present invention features methods, compositions, kits, and apparatus for determining the presence or absence of a target 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 target molecule and has the following formula:
5′—A—B—C—3′.
As used above, A is 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 RNA sequence, F, being replicated. The letter “B” denotes a section of the RNA molecule having approximately 10 to 50,000 nucleotides, which section is capable of binding to the target molecule. The letter “C” denotes a section of the RNA molecule having approximately 1 to 10,000 nucleotides which section is capable of being ligated to another RNA sequence, “D”. The second RNA molecule is capable of binding to a target molecule and has the following formula:
5′—D—E—F—3′.
As used above, D is a section of the RNA molecule having approximately 1 to 10,000 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 50,000 nucleotides, which section is capable of binding to the target molecule. 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 capable of forming a third RNA molecule having the following formula:
5′—A—B—C—D—E—F—3′.
The third RNA molecule is formed by ligation the C and D sections, as the E and the B sections are bound to the target. The third RNA molecule is capable of being received by an RNA replicase and being replicated by such enzyme.
Preferably, the sequences represented by the letters “A” and “F” are selected from the group of sequences consisting of MDV-I RNA, Q-beta RNA microvariant RNA, nanovariant RNA, midivariant RNA and modifications of such sequences that maintain the ability of the sequences to be replicated by RNA replicase. Preferably, the replicase is Q-beta replicase.
Preferably, the sections B and E each are sections having 10-5,000 nucleotides and, even more preferred, 20-50 nucleotides. Preferably, the sections B and E bind to the target through non-nucleic acid base pairing interactions. And, preferably, the B and E sections are aptamers or partial aptamers as defined by Klug and Famulok (1994). Aptamers are selected for a particular functionality, such as binding to small or large organic molecules, peptides or proteins, the tertiary structure of nucleic acids or complex or simple carbohydrates. The section B and E may be derived from naturally occurring RNA exhibiting affinity for proteins. The sections B and E may also be engineered from computer modeling studies.
Preferably, the sections C and D each have 1-10,000 nucleotides, and more preferred, 1-1000 nucleotides, and most preferred, 1-15 nucleotides. Preferably, the sections C and D, when ligated together, define a recognition site for a ribozyme or a target of another compound that has an endonucleolytic activity against a single-stranded nucleic acid.
A further embodiment of the present invention features a method of determining the presence or absence of a target molecule. The method comprises the steps of providing a first RNA molecule and a second RNA molecule. The first RNA molecule is capable of binding to a target molecule and has the formula:
5′—A—B—C—3′.
The sections A, B and C are as previously described. The second RNA molecule is capable of binding to the target molecule and has the formula:
5′—D—E—F—3′.
The sections D, E and F are as previously described. The method further comprises the step of imposing binding conditions on a sample potentially containing target molecules in the presence of the first and second RNA molecules. In the presence of the target molecule, the first and the second RNA molecules form a complex with the target molecule. The method further comprises the step of imposing RNA ligase reaction conditions on the sample to form a third RNA molecule in the presence of the target. The third RNA molecule has the formula:
5′—A—B—C—D—E—F—3′.
The sample is monitored for the presence of the third RNA molecule, presence or absence of which is indicative of the presence or absence of the target molecule.
Preferably, the sections B and E bind to the target through non-nucleic acid pairing interactions. And, most preferred, the B and E sections are aptamers or partial aptamers.
Preferably the sections C and D together comprise 5-15 nucleotides which define a site for a ribozyme or a target of another compound that has endonucleolytic activity against a single-stranded nucleic acid.
Preferably, at least one of the first or second RNA molecules has a signal generating moiety. After RNA ligase reaction conditions are imposed, the method preferably comprises the further step of separating or enzymatically destroying the RNA molecules unbound with the target molecule and bearing the signal generating moiety.
Preferably, the signal-generating moiety is sections A and F of the third RNA molecule, which sections allow recognition and replication by RNA replicase. Thus, the method further comprises the st
Houtteman Scott W.
InVitro Diagnostics, Inc.
Janiuk Anthony J.
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