Bis-locked nucleic acids : a new tool for double helix invasion

摘要

Deoxyribonucleic acid (DNA) is a macromolecule that contains the information for all livingorganisms to achieve important cellular processes such as growth and replication. DNAconsists of two strands coiled around each other to form a double helix. Several interactionsincluding Watson-Crick pairing and stacking between the bases stabilize the double helix. Toensure the integrity of genetic information, the bases are on the inside the double helixwhereas the phosphate and sugar groups are on the outside. Thus, most DNA information isnot accessible until DNA is unwound prior to replication or transcription. In cells, enzymescalled helicases achieve DNA strands separation.The anti-gene strategy aims to introduce a short single-stranded oligonucleotide (ON) to bindgenomic DNA at a specific site to block the mRNA transcription. As a result, mRNA andprotein expression for a specific gene are expected to be reduced, which could be beneficialin certain clinical contexts. However, this therapeutic strategy is hampered by the lowstability of the DNA binding, the difficulty to reach the genomic target and to block the RNApolymerase, suggesting the needs for new DNA binding ONs.In this thesis, we developed clamp-ONs, bis-locked nucleic (bisLNAs), binding viaHoogsteen and Watson-Crick interactions. In paper I, we assessed their bindingcharacteristics and demonstrated their ability to invade supercoiled DNA at intranuclear pHand salt conditions. In paper II, we investigated the step-by-step mechanism for bisLNAinvasion. Based on this mechanism, we designed a new generation of bisLNAs using nonnatural modifications such as a stacking linker and 2´-glycylamino LNAs. In paper III, weinvestigated how the bisLNAs invade in a more complex environment using rolling circleamplification for detection.