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Nucleic Acids Research, 1991, Vol. 19, No. 11 2993-3000
© 1991

MOLECULAR BIOLOGY

Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts

J. Schonborn, J. Oberstraß, E. Breyel1, J. Tittgen2, J. Schumacher2 and N. Lukacs*

Heinrich-Heine-Universitat Dusseldorf, Institut fur Physikalische Biologie Dusseldorf 1DSM-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Abteilung Pflanzenviren Braunschweig 2DIAGEN, Institut fur Molekulare Diagnostik GmbH Dusseldorf, FRG

*To whom correspondence should be addressed

Received February 13, 1991. Revised May 14, 1991. Accepted May 14, 1991.

We describe four monoclonal antibodies (MAB) which specifically recognize double-stranded RNA (dsRNA) together with their use in new methods for detecting and characterizing dsRNA in unfractionated nucleic acid extracts. The specificity of the antibodies was analyzed using a panel of 27 different synthetic and naturally occuring nucleic acids. All four antibodies reacted in a highly specific manner with long dsRNA helices, irrespective of their sequence; no binding to single-stranded RNA homopolymers or to DNA or RNADNA hybrids was observed. The apparent affinity of the antibodies to short (≤11bp) RNA helices was very low in all test systems used: only background levels of binding were obtained on single-stranded RNA species which contain double-helical secondary structures (e.g. rRNA, tRNA, viroid RNA). A sandwich ELISA and a dsRNA-immunoblotting procedure have been established which allow detection and characterization of dsRNA by MAB even in the presence of a large excess of other nucleic acids. In combination with temperature-gradient gelelectrophoresis (TGGE) not only the molecular weights but also the highly characteristic Tm-values of conformational transitions of individual dsRNA species could be determined by immunoblotting. An example of the general use of these methods for the detection of plant virus infections is demonstrated with groundnut rosette virus (GRV) dsRNAs. We were able to estimate the dsRNA content of infected leaves, identify the dsRNA species present in crude extracts and to determine the Tm-values of GRV dsRNA-3.


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