Clamp oligonucleotides inhibit DNA primer extension on a single-stranded template. They can also arrest reverse transcription on a single-stranded RNA template. Clamp oligonucleotides are useful tools for sequence-specific control of gene expression as therapeutic agents as well as BNA clamp probe-based real-time PCR foor the detection of mutated genes.
A clamp oligonucleotide is a single-stranded nucleic acid that a complementary (antisense) oligonucleotide can recognize to form a short double helix. A third clamp oligonucleotide can recognize an oligopyrimidine-oligopurine helical formation to form a triple helix. The two oligonucleotides can be linked to each other, creating a unique molecule that can clamp the target sequence on the single-stranded template (Giovannangeli et al. 1991). The nature of the third strand determines its orientation depending on the oligo target sequence. Therefore, the linker sequence between the antisense and the third nucleic acids strand portions will join a 3'-end to a 3'-end or a 5'-end to a 5'-end (resulting in an antiparallel orientation of the third strand), or a 3'-end to a 5'-end (resulting in a parallel orientation). In the latter case, a circular oligonucleotide can be synthesized (Kool 1991). For some entropic reasons, the circular oligonucleotide will bind more tightly than the clamp oligonucleotide, which in turn binds much more tightly than two separate oligonucleotides (at least in the micromolar range of concentrations).
Studies have shown that BNA clamp oligonucleotides can detect EGFR mutation with a mutated version of the target sequence using the same clamp oligonucleotide. Clamp oligonucleotides can be covalently attached to intercalating agents. If the antisense portion is somewhat longer than the third strand portion, intercalation can lock the complex in place on the single-stranded target (Giovannageli et al., 1993). BNA modified oligonucleotides form stable triple helices. These are alternative tools to antisense oligonucleotides for the control of gene function. A BNA antigene oligonucleotide targeted to the DNA double helix can inhibit transcription. Clamp oligonucleotides targeted to a viral sequence can inhibit reverse transcription. They can also impede the translation of a messenger RNA. Strand displacement reactions observed using BNAs (which involve triple helix formation by two BNAs on one of the two strands) represent an alternative to antigene oligonucleotides that bind to DNA without opening the double helix.
C Giovannangeli, N T Thuong, C Hélène; Oligonucleotide clamps arrest DNA synthesis on a single-stranded DNA target.Proceedings of the National Academy of Sciences Nov 1993, 90 (21) 10013-10017. [PNAS]