Abstract
Antisense oligonucleotides bind to their complementary sequence on messenger RNAs and inhibit translation of the mRNA into the protein. Short oligonucleotides may also be used to inhibit gene transcription via triple-helix formation on a double-stranded DNA gene. These oligonucleotides are called triplex-forming oligonucleotides (TFO). Certain TFOs (also called clamp oligonucleotides) are designed to bind to single-stranded nucleic acids and may inhibit mRNA translation by physical blockade. The numerous applications of triplex-based strategies have been well described. Triplex-forming oligonucleotides and antisense oligonucleotides share the same requirements for cellular applications, such as high binding affinity for their targets, nuclease resistance, and efficient cell penetration. Very similar physicochemical methods are used to study antisense and triplex-forming oligonucleotides in vitro before considering their utilization in cell culture. This chapter focuses on some of the methods employed to determine the critical parameters that help in designing an efficient antisense or triplex-forming oligonucleotide.
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