Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1995-06-07
2002-07-16
Horlick, Kenneth R. (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S022100, C536S024310, C435S091100, C435S091200
Reexamination Certificate
active
06420539
ABSTRACT:
BACKGROUND OF THE INVENTION
Throughout this application, various publications are referenced and citations provided for them. The disclosure of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
It is now a well established fact that all living organisms including infectious agents, e.g., viruses, contain DNA, or sometimes RNA, molecules which carry genetic information in the form of a nucleotide sequence code. While certain segments of this code are shared by many organisms, there are other segments which contain nucleotide sequences that are unique for a particular organism. These sequences are said to be species-specific and provide a convenient tag or footprint that can be utilized for identification of that organism. The technique of nucleic acid hybridization (Gillespie and Spiegelman, 1965) has great potential for the rapid detection and typing of infectious agents. However, current hybridization assays have not yet attained the sensitivity and speed required for practical diagnostic use. It has recently been proposed that the sensitivity and speed of bioassays could be improved by linking a replicatable RNA to a hybridization probe (Chu, et al. 1986). After hybridization, the replicatable RNA would be amplified by incubation with the RNA-directed RNA polymerase, Q&bgr; replicase (Haruna and Spiegleman, 1965a). The enormous number of RNA copies that would be synthesized would serve as a signal that hybridization had occurred. The synthesis of novel nucleic acid hybridization probes that combine in a single RNA molecule the dual functions of probe and amplifiable reporter is described in this invention.
A distinguishing feature of RNA synthesis by Q&bgr; replicase is that a small number of template strands can initiate the synthesis of a large number of product strands (Haruna and Spiegleman, 1965b). Million-fold increases in the amount of RNA routinely occur in vitro (Kramer, et al. 1974) as a result of an autocatalytic reaction mechanism (Weissman, et al. 1986; Spiegelman, et al. 1968): single-stranded RNAs serve as templates for the synthesis of complementary single-stranded products; after the completion of product strand elongation, both the product and the template are released from the replication complex (Dobkin, et al. 1979); and both strands are free to serve as templates in the next round of synthesis. Consequently, as long as there is an excess of replicase, the number of RNA strands increases exponentially. After the number of RNA strands equals the number of active replicase molecules, RNA synthesis continues linearly.
Q&bgr; replicase was first isolated from bacteriophage Q&bgr;-infected
Escherichia coli
by Haruna and Spiegelman (1965a). It is composed of four polypeptides, only one of which is specified by the viral RNA. The other three polypeptides are
E. coli
proteins, and have been identified as the protein synthesis elongation factors Tu and Ts and the ribosomal protein S
1
. When provided with the single-stranded RNA from Q&bgr;, the replicase mediates the exponential synthesis of infectious viral RNA (Spiegelman et al., 1965). The enzyme is highly template selective. No other viral RNA, nor any
E. coli
RNA, will serve as a template (Haruna and Spiegelman, 1965c). When RNA from a temperature-sensitive mutant of Q&bgr; was used as a template with wild-type replicase, mutant RNA was synthesized, demonstrating that the template is the instructive agent (Pace and Spiegelman, 1966). The replicative process (Spiegelman et al., 1969; Weissmann et al., 1968) proceeds in the following manner: The replicase uses the viral (+) strand as a template to direct the synthesis of a complementary (−) strand. Both of these strands serve as templates for the synthesis of additional (+) and (−) strands; and exponential increase is observed in the number of RNA strands present. Eventually, there are enough strands to saturate the available enzyme molecules, after which the number of strands increases linearly with time. Because of the complementary nature of this process, it is often referred to as “self-replication”. There are a number of advantages to using the amplification of RNA by Q&bgr; replicase as the basis of a signal-generating system: Q&bgr; replicase is highly specific for its own template RNAs (Haruna and Spiegelman, 1965c); as little as one molecule of template RNA can, in principle, initiate replication (Levisohn and Spiegelman, 1968); and the amount of RNA synthesized (typically, 200 ng in 50 &mgr;l in 15 minutes) is so large that it can be measured with the aid of simple colorimetic techniques. There are a number of naturally occurring Q&bgr; replicase templates that are much smaller than Q&bgr; RNA. These RNAs have been isolated from in vitro Q&bgr; replicase reactions that were incubated in the absence of exogenous template RNA. They include: MDV-1 RNA (Kacian et al., 1972), microvariant RNA (Mills et al., 1975), the nanovariant RNAs (Schaffner et al., 1977), RQ120 RNA (Munishkin at al., 1989), and cordycepin-tolerant RNA (Priano et al., 1989). Although the origin and biological role of these RNAs is not known, they have been extensively characterized and are all excellent templates for Q&bgr; replicase.
Isolated MDV-1 RNA serves as an excellent exogenous template. It is bound by Q&bgr; replicase and replicated in a manner similar to Q&bgr; RNA (Kacian et al., 1972). MDV-1 RNA is much smaller (221 nucleotides) than Q&bgr; RNA (4,220 nucleotides), which led to the determination of its complete nucleotide sequence (Mills et al., 1973; Kramer and Mills, 1978).
Two striking aspects of the MDV-1 sequence are its unusually high proportion of guanosine and cytidine residues and the occurrence of many intrastrand complements capable of forming hairpin structures. MDV-1 has been directly visualized (Klotz et al., 1980), utilizing hollow-cone, dark-field electron microscopy. Observations made with native, partially denatured, and fully denatured molecules indicate that native single-stranded MDV-1 RNA is a highly condensed molecule, possessing substantial tertiary structure. Specific secondary structures were identified by reacting MDV-1 RNA with chemical agents that modify single-stranded regions (Mills et al., 1980). The location of the altered nucleotides was determined by sequencing the modified RNA. The tertiary structure of MDV-1 RNA was probed by subjecting it to mild cleavage with ribonuclease T
1
(Kramer et al., 1989), which only cleaves single-stranded regions. Because of the extensive secondary and tertiary structure present in MDV-1 RNA, combined with the macromolecular dimensions of ribonuclease T
1
, the initial sites of attack were limited to those on the exterior of the molecule. The few guanosines in each strand that were hypersusceptible to ribonuclease T
1
were located in hairpin loops.
A more detailed understanding of the mechanism of MDV-1 RNA synthesis was facilitated by the development of an electrophoretic technique for separating the complementary strands (Mills et al., 1978). An excess of pure MDV-1 (−) RNA was used as template in the presence of a small amount of Q&bgr; replicase, in a series of experiments designed to elucidate the synthetic cycle. Mutant MDV-1 (−) RNA (Kramer et al., 1974) was added to these reactions after the initiation of chain elongation to see whether replicase molecules retain the same template through many rounds of synthesis. It was shown that a single replicase molecule bound to a single template strand is sufficient to carry out a complete synthetic cycle. It was shown that after the completion of each round of chain elongation, the product strand is released from the replication complex, and the template and the replicase then dissociate (Dobkin et al., 1979).
Specific regions of MDV-1 RNA are required by Q&bgr; replicase to carry out different replication functions. A highly structured region in the middle of each complementary strand must
Kramer Fred Russell
Lizardi Paul M.
Miele Eleanor Ann
Mills Donald R.
Cooper & Dunham LLP
Horlick Kenneth R.
The Trustees of Columbia University in the City of New York
Tung J.
White John P.
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