17 - Nucleic Acid and Oligonucleotide Modification and Conjugation

Abstract

This chapter describes the major enzymatic and chemical modification procedures used to label nucleic acids and oligonucleotides. Radiolabeled probes are being replaced by molecular biology techniques utilizing nonradioactive detection for assaying oligonucleotide interactions. A prime factor in the development of nonradioactive systems for RNA and DNA measurement is the ability to modify a nucleic acid with a detectable component while not affecting base-pairing. The attachment of a small probe such as a fluorescent molecule or a large catalytic enzyme to an oligonucleotide forms the basis for constructing a sensitive hybridization reagent. The methods developed to cross-link or label proteins are not always applicable to nucleic acids. The main reactive sites on proteins involve primary amines, sulfhydryls, carboxylates, or phenolates—groups that are familiar and relatively easy to derivatize. RNA and DNA contain none of these functional groups. They also do not react with many of the common bioconjugate reagents. To modify the unique chemical groups on nucleic acids, novel methods have been developed that allow derivatization through discrete sites on the available bases, sugars, or phosphate groups. These chemical methods can be employed to add a functional group or a label to an individual nucleotide or to one or more sites in oligonucleotide probes or full-sized DNA or RNA polymers. If an individual nucleotide is modified in the appropriate way, various enzymatic techniques can be used to polymerize the derivative into an existing oligonucleotide molecule. Alternatively, nucleotide polymers can be treated with chemical activators that can facilitate the attachment of a label at particular reactive sites. Thus, there are two main approaches to modifying DNA or RNA molecules: enzymatic and chemical.