Theodore W. Munns

Antibodies Specific for Modified Nucleosides: An Immunochemical Approach for the Isolation and Characterization of Nucleic Acids

Publisher Summary The chapter discusses various antibodies specific for modified nucleosides that is an immunochemical approach for the isolation and characterization of nucleic acids. The rationale of an immunochemical approach for the isolation and characterization of nucleic acids is based on two important observations. First, most nucleic acids contain a limited number of modified nucleosides that are uniquely distributed throughout their structures. Second, antibodies that possess high degrees of affinity and specificity toward these modified nucleosides can be prepared, with minimal or no significant cross-reactivity, with the bulk of unmodified constituents present in nucleic acids. From the information presented in these sections, the feasibility of an immunochemical approach is demonstrated, by using specific anti-nucleoside antibodies: (a) to isolate hapten-containing oligonucleotides and ribonucleic acids (RNAs), (b) to assess the functional significance of the hapten component, and (c) to map the position of the hapten, within selected nucleic acid structures. The employment of the anti-nucleoside antibody is by no means restricted to the naturally occurring modified constituents of nucleic acids. Equally or perhaps more significant are their application in the detection, quantitation, isolation, and location of aberrant nucleoside adducts that appear in the nucleic acids of organisms exposed to various forms of radiation.

Role of N6-Methyladenosine in Expression of Rous Sarcoma Virus RNA: Analyses Utilizing Immunoglobulin Specific for N6-Methyladenosine

Publisher Summary Antiserum specific for N 6 -methyladenosine has been generated and well characterized This chapter explores the use of such an immunoglobulin preparation (derived by ammonium sulfate precipitation) in isolation of specific m 6 A-containing regions of Rous sarcoma virus RNA and its use as a site-specific probe in the analysis of m 6 A function in RNA. When purified 38 S genome RNA from RSV is incubated with RNase-free anti-m 6 A immunoglobulin, a substantial fraction (>60%) of the total RNA may be recovered by binding to Sepharose-protein A. When viral RNA is fragmented (200-2000 nucleotides), the binding is significantly decreased. When the RNA is fragmented even more extensively by digestion with ribonuclease T1, the percentage of RNA capable of binding Sepharose-protein A is diminished to 1-2% of the total mass. The specificity of the binding reaction is also demonstrated by the fact that prior incubation of the immunoglobulin with free m 6 A completely inhibits the binding of viral RNA.