Targeting Degradation of RNA by RNase H Using DNA Hairpins

Abstract

RNase H degradation of two 15 nt RNA target sites was examined in the presence of hairpin DNAs with a 5 nt loop and a 10 bp stem or single-stranded 15 nt DNAs. One target site was a segment of a 79 nt RNA, and the other was part of a 53 nt RNA. Secondary structure predictions indicate that the 53 nt RNA target site is entirely single stranded, while a portion of the 79 nt RNA target site forms an intramolecular duplex. Less RNase H and DNA were needed to cleave the 53 nt RNA target site than the less accessible 79 nt RNA site. The hairpin DNAs had their 5 nt loop and 3' side of the stem fully complementary to the target sites or had sequence changes that produced one to nine mismatched pairs. T(m) values ranged from 57 to 80 degrees C. The stability of the hairpin DNAs relative to the stability of their corresponding RNA-DNA hybrids influenced the extent of RNase H degradation at 37 degrees C. Under the assay conditions employed, the amount of degradation directed by the hairpin DNAs was correlated with their predicted DeltaG(o) (37) of binding to the RNA targets. A DNA hairpin with one mismatch to the target site of the 79 nt RNA did not induce degradation under conditions where fully complementary DNA hairpins produced 50-80% degradation. The in vitro results indicate that DNA hairpins can enhance the stringency of RNase H targeted degradation of the RNA sites.

Thermal stability, structural features, and B‐to‐Z transition in DNA tetraloop hairpins as determined by optical spectroscopy in d(CG)3T4(CG)3 and d(CG)3A4(CG)3 oligodeoxynucleotides

“…Moreover, folded regions of nucleic acids are their most exposed parts to the solvent and surrounding molecules, explaining the privileged role of DNA (or RNA) hairpins in intermolecular recognition and interactions with regulatory proteins and enzymes 14, 15. On the other hand, in the framework of gene therapy, antisense oligodeoxynucleotides have been currently conjugated to ultrastable hairpins in order to increase their stability against cellular nucleases 16, 17…”

Mouse Retina Has Oligonucleotide-Induced Gene Repair Activity

“…Such an effect may have detracted from repair of the tet gene by RDOTET1. In addition, because fully self-complementary RDOs (e.g., RDOTET1) are expected to have significantly higher melting temperatures than RDOs with an internal mismatch 31 (e.g., RDOTET2), it should be more energetically favorable for RDOTET2 than RDOTET1 to participate in heteroduplex formation with the target DNA. Consequently, a possible increase in heteroduplex formation may have contributed to the increased repair observed with RDOTET2.…”

Regulating gene expression with light-activated oligonucleotides

“…Cell stability can be enhanced considerably by modifying the backbone of the oligonucleotide, as exemplified by phosphorothioate, 69 peptide, 61 morpholine, 59 and 'locked' 70 nucleic acids. Light-activated methods for controlling the binding of antisense oligodeoxynucleotides (asODNs) to target mRNA would permit the reduction of protein expression in a cell-or tissue-specific fashion, by either sterically blocking the ribosome 59,60 or recruiting ribonucleases [71][72][73] that bind specifically to DNA/RNA duplexes. asODNs have proven effective in gene silencing in many experimental systems, 71,[74][75][76] and are being evaluated as treatments for cancer and other diseases in human clinical trials.…”