Method for monitoring transcriptional synthesis of RNA

Details Method for monitoring transcriptional synthesis of RNA Method for monitoring transcriptional synthesis of RNA

1999-04-02

2001-01-30

Horlick, Kenneth R. (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091300, C435S091500

Reexamination Certificate

active

06180347

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
This invention relates to a method for monitoring the transcriptional synthesis of RNA and to a device therefor.
BACKGROUND OF THE INVENTION
Proteins are synthesized on ribosomes based on the base sequences of RNA that are transcribed from those of DNA. Transcription reaction is the first stage toward protein synthesis and refers to a process that transcribes base sequences of DNA present within cell nuclei and synthesizes RNA. Specifically, it is a reaction that synthesizes complementary RNA from DNA, which serves as a template (i.e., adenine to uracil, or guanine to cytosine), in the 5′- to 3′-direction.
After RNA polymerase that is responsible for RNA synthesis has bound to a specific base sequence on DNA which is referred to as “promoter,” it incorporates nucleotide triphosphates (NTP), which are complementary to the DNA bases, from the site of transcription initiation on the template DNA and it synthesizes RNA. Polymerization proceeds a few nucleotides away from the site of transcription initiation; and then, stable polymerization continues and the RNA strand is further elongated, separating from its DNA pairing. This series of polymerization reactions continues until a specific terminator sequence on the DNA appears.
In vitro transcription reaction is a process that utilizes the transcription mechanism in the cells and synthesize RNA in vitro with simplicity, and it requires RNA polymerase, cofactors thereof, NTP substrates, and template DNA.
In recent years in vitro transcription reactions utilizing T7, T3 and SP6 polymerases, which do not require the transcription cofactors, have been in frequent use. The in vitro transcription reaction utilizing T7, T3, or SP6 polymerase is a method to synthesize the desired RNA strand: it introduces a DNA fragment, which will serve as the template, into the downstream of each promoter, and carries out a transcription reaction by utilizing an RNA polymerase that specifically recognizes the promoter.
The in vitro transcription reaction allows the synthesis of RNA into which a cap structure or an intron is introduced in addition to that of long-strand RNA, both of which are difficult to achieve through automated synthesizers. These RNAs are in wide use as template RNA for in vitro translation or as probes for hybridization. The RNA to which a cap structure has been appended or that into which an intron has been introduced is used to analyze the termini or intervening sequences of transcripts. (Current Protocols in Molecular Biology, Green Publishing Associates, Inc. and John Wiley & Sons, Inc. 1996.)
Thus, in vitro transcription reactions for RNA synthesis are utilized in many research fields. However, a variety of conditions generally need to be precisely satisfied in order to allow an in vitro transcription reaction to proceed normally: among others, a promoter is to be incorporated into a suitable site on the DNA that is used as a template and the combination of the promoter and RNA polymerase must be appropriate. Also, for the purpose of carrying out the transcription reaction efficiently in a prepared sample, there is a need for setting detailed conditions, such as the quantity ratio of the template DNA to the RNA polymerase, to optimum values. Whether the sample prepared to carry out the in vitro transcription reaction satisfy the conditions can not be known until the reaction is actually conducted and the result is analyzed to determine whether the desired product has been produced. In practice, after the reaction solution is sampled at predetermined intervals and RNA is extracted, the absorption value at 260 nm is measured: this confirms that the quantity of the synthesized RNA in the reaction solution has increased, which is routinely used to determine whether the reaction has proceeded. Further, to ascertain that the synthesized RNA is of full-length, the determination is routinely made by the electrophoresis method after the RNA is extracted. These methods require manipulations for extracting the synthesized RNA, and the procedure therefor is normally very complicated and also time-consuming. Thus, the procedure must be repeated to determine conditions that allow the in vitro transcription reaction to proceed efficiently without irregularities, which requires a great amount of time and labor.
Known as a method for the detection of a nucleic acid such as single-stranded DNA is that which utilizes two types of fluorescence-labeled probes that hybridize with said nucleic acid. This comprises adding to a solution containing the nucleic acid, two types of probes labeled with fluorescent dyes that differ from each other in their kinds; and it takes advantage of the fact that when the two types of probes hybridize to the same nucleic acid adjacently to each other, the distance between the fluorescent dyes becomes shorter and thus, resonance energy transfer occurs between the fluorescent dyes with the result of changes in fluorescence spectra. (Cardullo, R. A. et al. (1988) Proc. Natl. Acad. Sci. USA. 85, 8790-8794 and U.S. Pat. No. 4,996,143 (Heller et al.)). When this method is applied to the detection of single-strand RNA, it is necessary that any site (a base sequence) on the RNA to which the probe can hybridize be searched in advance through experiments. Generally, RNA having not less than tens of bases in length adopts a specific stereostructure (secondary structure) in aqueous solution, and as a result, most sites in the RNA do not undergo hybridization with other nucleic acids. (Chiang, M. -Y., Chan H. et al., J. Biol. Chem. 266, 18162-18172, (1991).) Techniques for determining the sites on RNA to which probes can hybridize with reliability have not been established. Generally, the secondary structure of the RNA is simulated by computer (Michael Zucker and Patrick Stiegler, Nucleic Acid Research, 9, 133-148, 1981); in the predicted structure, candidates for the site to which the probe can hybridize are picked up; probes having their base sequences are prepared; and the presence or absence of hybridization between the respective probes and the RNA is determined experimentally. These manipulations are very complicated and need a great amount of time as well. Also, even if these manipulations are repeated, it is not necessarily guaranteed that any site that will hybridize to said RNA can be located. For these reasons, there has been no report of the case where RNA having not less than 100 bases in length was detected according to said method.
BRIEF SUMMARY OF THE INVENTION
This invention has remedied the above-mentioned drawbacks and provides a method for monitoring a transcription reaction for RNA synthesis on time without requiring either manipulations for RNA extraction or operations in electrophoresis. Specifically, it provides a method for monitoring the initiation, the progression, and the termination of the transcription reaction for RNA synthesis, as well as the quantitation of the total RNA to be synthesized by the method described above and the synthesis of full-length RNA. Further, it provides a method capable of measuring reaction rates with regard to the transcription reaction (the speed of rotation of the transcription) and of optimizing conditions for the reactions described above.
Furthermore, this invention provides a monitoring apparatus relying on the methods.


REFERENCES:
patent: 4996143 (1991-02-01), Heller et al.
patent: 5814447 (1998-09-01), Ishiguro et al.
patent: WO 95/03428 (1995-02-01), None
patent: WO 98/13524 (1998-04-01), None
Amersham Life Science catalog, “Multicolor Fluorescent Reagents and Directly Labeled Biologicals” (date unknown).
Brown et al., “Modern machine-aided methods of oligodeoxyribonucleotide synthesis,”in Practical Approach Series: Oligonucleotides and Analogues(Eckstein F, ED.), pp. 1-24 (IRL Press at Oxford University Press, Oxford, England (ISBN 0-19-963279-0) 1991).
Cardullo et al.,Poc. Natl. Acad. Sci. USA, 85, 8790-8794 (Dec. 1988).
Chiang et al.,The Journal of Biological Chemistry, 266 (27), 18162-18171 (Sep. 25, 1991).
“DNA-Dep

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