Deoxyribonucleic acid (DNA) is a macromolecule that contains the information for all living
organisms to achieve important cellular processes such as growth and replication. DNA
consists of two strands coiled around each other to form a double helix. Several interactions
including Watson-Crick pairing and stacking between the bases stabilize the double helix. To
ensure the integrity of genetic information, the bases are on the inside the double helix
whereas the phosphate and sugar groups are on the outside. Thus, most DNA information is
not accessible until DNA is unwound prior to replication or transcription. In cells, enzymes
called helicases achieve DNA strands separation.
The anti-gene strategy aims to introduce a short single-stranded oligonucleotide (ON) to bind
genomic DNA at a specific site to block the mRNA transcription. As a result, mRNA and
protein expression for a specific gene are expected to be reduced, which could be beneficial
in certain clinical contexts. However, this therapeutic strategy is hampered by the low
stability of the DNA binding, the difficulty to reach the genomic target and to block the RNA
polymerase, suggesting the needs for new DNA binding ONs.
In this thesis, we developed clamp-ONs, bis-locked nucleic (bisLNAs), binding via
Hoogsteen and Watson-Crick interactions. In paper I, we assessed their binding
characteristics and demonstrated their ability to invade supercoiled DNA at intranuclear pH
and salt conditions. In paper II, we investigated the step-by-step mechanism for bisLNA
invasion. 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, we
investigated how the bisLNAs invade in a more complex environment using rolling circle
amplification for detection