Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-03-26
2002-07-09
Horlick, Kenneth R. (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06416951
ABSTRACT:
The present application concerns methods for the identification of functional antisense agents. In particular, the present application is concerned with improved methods for measuring the kinetics of the hybridisation of RNA and antisense oligonucleotides.
WO 98/15651 provides methods to identify regions in the primary sequence of a target RNA that are accessible to hybridisation probes. The methods involve hybridising short oligonucleotide probes to the target RNA in separate reaction zones. The sequences of the hybridising probes are compared with the primary sequence of the target RNA to identify probes which hybridise to overlapping sequences in the target RNA. These sequences identify accessible regions in the target RNA. WO 98/15651 further describes the use of radiolabelling as an embodiment of the invention in which the hybridisation reactions between the probes of that invention and a target RNA can be followed in real time.
It is an object of this invention to provide non-radioactive methods for following the hybridisation reactions described in WO 98/15651 in real time. It is also object of this invention to provide compounds and kits to practice the methods of WO 98/15651. The methods of RNA analysis described in this prior patent application are based on hybridising an array of oligonucleotides to a target RNA. In the earlier application the hybridisation reactions between each member of the array and the target RNA are performed in spatially separated reaction vessels. It is a further object of this invention to provide methods of analysing a target RNA that generate the same data as the methods of the previous disclosure without requiring complete spatial separation of all of the probes in the array used to analyse the target RNA.
Accordingly, the present invention provides a method for identifying a functional antisense agent, which method comprises hybridising an RNA with an oligonucleotide probe and measuring in real time the kinetics of hybridisation, wherein the kinetics are measured by either hybridising in the presence of an intercalation dye and recording a change in the spectroscopic properties of the dye as hybridising proceeds, or incorporating a label in either the RNA or the probe, attaching the non-labelled RNA or non-labelled probe to a solid support, generating an evanescent wave in the proximity of the non-labelled RNA or non-labelled probe and recording the increase in a signal generated by interaction of the evanescent wave with the label, as hybridisation proceeds, and wherein the oligonucleotide probe comprises an array of oligonucleotides, each oligonucleotide in the array having a common length of 4 to 8 nucleotides, all possible base sequences of that length being represented in the array.
This invention also provides a kit for identifying a functional antisense agent, comprising an oligonucleotide probe, and a means for generating a signal for measuring the kinetics of hybridisation of the oligonucleotide probe to an RNA, wherein the oligonucleotide probe comprises an array of oligonucleotides, each oligonucleotide in the array having a common length of 4 to 8 nucleotides, all possible base sequences of that length being represented in the array.
Detection using evanescent waves
A non-radioactive method to determine the kinetics of hybridisation reactions of the sort described in WO 98/15651 in real time would be preferable to radiolabelling for safety reasons. A method of measuring the kinetics of nucleic acid hybridisation is described by D. I. Stimpson et al in Proc. Natl. Acad. Sci. USA, 92: 6379-6383, 1995. The method disclosed in this publication measures the hybridisation of a labelled oligonucleotide probe to a target immobilised at the surface of a two dimensional optical wave guide. An optical wave guide that is transmitting light generates an>evanescent wave×which extends some distance above the surface of the wave guide. Light from the evanescent wave can be scattered by a particulate label near the surface of the wave guide. This scattered light can be detected by a Charge Coupled Device camera, a fluorescent microscope or any other appropriate optoelectronic detection instrument. The quantity of scattered light is a measure of the quantity of particulate label near the surface of the wave guide. The evanescent wave extends only about 100 to 300 nm beyond the surface of the wave guide so only labelled material close to the surface of the wave guide will scatter light. This is ideal for measuring binding, unbinding or cleavage events close to the surface of the wave guide as a binding reaction at the surface of the wave guide will greatly increase the quantity of labelled probe within the evanescent wave over the background of label present free in solution. If a target RNA molecule is labelled with an appropriate particulate label the methods of the above publication could be applied with the methods of this invention. In the above publication by Stimpson et al, 200 nm selenium particles were conjugated with an anti-biotin antibody and linked to biotinylated oligonucleotide probes. Biotinylation of one terminus of a target RNA would allow a similar label to be used with this invention.
REFERENCES:
patent: 5750337 (1998-05-01), Squirrell
patent: WO-95/21265 (1995-08-01), None
patent: WO97/27331 (1997-07-01), None
patent: WO98/15651 (1998-04-01), None
Yguerabide, J. et al., “Quantitative Fluorescence Method for Continuous Measurement of DNA Hybridization Kinetics Using a Fluorescent Intercalator”, Anal. Biochem. vol. 228, pp. 208-220 (1995).*
Stimpson, D.I. et al., “Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides”, PNAS USA, vol. 92, pp. 6379-6383 (1995).*
Stimpson, Don et al., “Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides”Proc. Natl. Acad. Sci. USA,vol. 92, (Jul. 1995) pp. 6379-6383.
Schwille, Petra et al., “Quantitative Hybridization Kinetics of DNA Probes to RNA in Solution Followed by Diffusional Fluorescence Correlation Analysis”Biochemistry,vol. 35 (1996) pp. 10182-10193.
Ishiguro, Takahiko et al., “Fluorescence detection of specific sequence of nucleic acids by oxazole yellow-linked oligonucleotides. Homogeneous quantitative monitoring of in vitro transcription”Nucleic Acids Research,vol. 24, No. 24 (1996) pp. 4992-4997.
Abstract—Yguerabide, J. et al., “Quantitative fluorescence method for continuous measurement of DNA hybridization kinetics using a fluorescent intercalator”,Analytical Biochemistry,vol. 228, No. 2, (Jul. 1995) pp. 208-220.
Abel, Andreas et al., “Fiber-Optic Evanescent Wave Biosensor for the Detection of Oligonucleotides”Anal. Chem.,vol. 68 (1996) pp. 2905-2912.
Bier, Frank F. et al., “Real-time measurement of nucleic-acid hybridization using evanescent-wave sensors: steps towards the genosensor”Sensors and Actuators B.38-39 (1997) pp. 78-82.
Schmidt Gunter
Thompson Andrew Hugin
Brax Group Ltd.
Horlick Kenneth R.
Strzelecka Teresa
Teskin Robin L.
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