Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-01-13
2001-05-01
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S024300, 19
Reexamination Certificate
active
06225058
ABSTRACT:
FIELD OF 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 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, are labor intensive and have low sensitivity (Nakamura 1993).
There exists a need to perform analytical and diagnostic assays of high sensitivity and high specificity. There exists a need for analytical methods, compositions and devices which facilitate the performance of a analytical or diagnostic procedure in less than one hour. There exists a need for analytical methods, compositions and devices which are directed to targets which are present in cells in quantities greater than one to one thousand copies. There exists a need for analytical and diagnostic procedures which identify small or large organic molecules, peptides or proteins, the tertiary structure of nucleic acids or complex or simple carbohydrates.
SUMMARY OF THE 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. The first RNA molecule binds a target molecule and has the following formula:
5′-A-B-C-D-E-3′.
As used above, A is a section of the RNA molecule having 10-100,000 nucleotides which section is, with another RNA sequence, E, replicated by an RNA replicase. The letter “B” denotes a section of the RNA molecule having approximately 1 to 50000 nucleotides which section, with another sequence D, binds the target molecule under binding conditions. The letter “C” denotes a section of the RNA molecule having approximately 1 to 10000 nucleotides which section is capable preventing the replication of the first molecule by the RNA replicase. The letter “D” denotes a section of the RNA molecule having approximately 1 to 50000 nucleotides which section, with another sequence B, binds the target molecule under binding conditions. The sections B and D, in combination, comprise in total at least 10 nucleotides. The first RNA molecule, with sections B and D bound to target, is acted upon by the RNA replicase to form a second RNA molecule. The second RNA molecule has the following formula:
5′-E′-X-A′-3′.
As used above, E′ is the complement to E, and A′ is the complement to A. The letter “X” denotes the complement of parts of the sections B, and D which may be replicated, or the letter denotes the direct bond between sections E′ and A′. The second RNA molecule is replicated by the RNA replicase under replicating conditions.
Preferably, the sequences represented by the letters “A” and “E” are selected from the group of sequences consisting of MDV-I RNA, Q-beta RNA microvariant RNA, nanovariant RNA, midivariant RNA, RQ-135 and modifications of such sequences which maintain the ability of the sequences to be replicated by Q-beta replicase. Preferably, the replicase is Q-beta replicase.
Preferably, the sections B and D have a combined total of 20-5,000 nucleotides and, even more preferred, 20-50 nucleotides. Preferably, the sections B and D bind to target through non-nucleic acid base pairing interactions. Sections B and D bind to the target in the manner of naturally occurring nucleic acid which form RNA-protein complexes. Or, the B and D sections are non-naturally occurring sequences which are selected to bind the target. These non-naturally occurring sequences are selected by computer modeling, or aptamers or partial aptamers, and other nucleic acids exhibiting affinity to the target. The term “aptomer” is used in the manner of Klug, S. J. and Famulok, M. “All you wanted to know about SELEX”, Molecular Biology Reports, 20:97-107 (1994) and other nucleic acids which are selected for affinity to a selected target. 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.
Preferably, the section B has a hybridization sequence of 1-100, and more preferred, 1-50, and most preferred, 1-5 nucleotides adjacent to the section A which form a hybridization product with a complementary hybridization sequence of section D. The nucleotides of the hybridization sequence of section D are adjacent section E. The hybridization sequences of sections B and D preferably define a loop or hairpin at such times that section B and D are bound to target. In the absence of target, the hybridization sequences do not form a stable hybridization product. In the presence of the target, and the formation of a complex between sections B and D with the target, a hybridization product is formed that allows the RNA replicase to skip sections B, C and D and replicate sections A and E.
Preferably, X comprises less than five nucleotides of sections B and D, and the second molecule resembles a wild-type template.
Preferably, the section C has 1-10,000 nucleotides, and more preferred, 1-1000 nucleotides, and most preferred, 1-100 nucleotides which sequences define a stop sequence for the RNA replicase. Stop sequences comprise one or more sequences which the RNA replicase can not read through to effect replication of the sequence. These sequences include, by way of example, without limitation, a sequence of poly A, poly C, poly G, multiple initiation sites, modified nucleotides which do not allow the RNA replicase to act on the sequence, sugar linkages without nucleotides and altered phosphate or sugar linkages.
Preferably, the sections A and E comprise at least one sequence that hybridizes to a third nucleic acid. Such third nucleic acid forms a hybridization product which hybridization product can be detected by known means.
A second embodiment of the present invention features paired RNA molecules comprising a first RNA molecule. The first RNA molecule binds a target molecule and has the following formula:
5′-A-F-B-3′.
And, the second RNA binds the target and has the following formula:
5′-D-H-E-3′
As used above, A is a section of the RNA molecule having 10-100,000 nucleotides which section is, with another RNA sequence, E, replicated by an RNA replicase. The letter “B” denotes a section of the RNA molecule having approximately 1 to 50000 nucleotides which section, with another sequence D, binds the target molecule under binding conditions. The letter “D” denotes a section of the RNA molecule having approximately 1 to 50000 nucleotides which section, with another sequence B, binds the target molecule under binding conditions. The sections B and D, in combination, comprise in total at least 10 nucleotides. The letter “F” denotes a section of the RNA molecule having has a hybridization sequence of 1-10,000, and more preferred, 1-50, and most preferred, 1-5 nucleotides which form a hybridization product with a complementary hybridization sequence of section H. The letter “H” denotes
Grossman Abraham
Munishkin Alexander
Chakrabarti Arun Kr.
Fredman Jeffrey
InVitro Diagnostics, Inc.
Janiuk Anthony J.
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