10,11,12,13 RNAs comprised of expanded CUG, CAG, CCG, CGG, GAA, or CCUG repeats can bind to TNF-�� inhibitor the template strand of DNA, forming RNA:DNA hybrids (R-loops).14,15,16,17 Whether these R-loops result from incomplete dissociation of nascent transcripts from template, or reassociation of transcripts with template, is unclear. Either way, the presence of R-loops may instigate the formation of extrahelical slipped strand structures on the nontemplate strand. Recognition of these structures by mismatch repair proteins may stimulate error-prone repair, leading to further expansion. A second unusual feature of the DM1 mutation is that it leads to the production of a toxic RNA.18,19 Interactions of CUGexp RNA with proteins are thought to underlie many symptoms of the disease.
One approach for therapy has focused on CAG-repeat antisense oligonucleotides (ASOs) designed to bind CUG repeat RNA, thereby blocking RNA-protein interactions by steric inhibition.20,21 By this strategy the activity of RNA binding proteins and regulation of alternative splicing are normalized in mouse models of DM1.20 Unexpectedly these ��blocker �� ASOs, although not competent to activate RNase H, were shown to reduce the level of CUGexp transcripts.20,21 Although the mechanism for this effect has not been determined, evidence suggests that release of CUGexp transcripts from nuclear foci can facilitate their transport to the cytoplasm, where they may undergo more rapid decay. While therapeutic efforts have focused on using ASOs to reverse RNA toxicity in DM1, the potential impact on DNA instability has not been addressed.
If R-loops at the DM1 locus result partly from reassociation of CUG repeats with template, then increased nuclear export or knockdown of CUGexp transcripts may inhibit this process and stabilize the expanded repeat. By contrast, CAG-repeat ASOs designed to bind toxic RNA may also hybridize to the nontemplate strand of DNA (CTG-repeat strand), forming D-loops that could, in theory, increase the instability of expanded repeats. If this occurs, the possibility exists that continued growth of the expanded repeat may ultimately defeat the therapeutic effect, due to increased length and toxicity of the CUGexp RNA. Here we used human cells and transgenic mice to examine the effects of CAG-repeat ASOs on instability of expanded CTG?CAG repeats.
Results Generation of CUG-expressing cells that have free uptake of ASOs In previous studies, we found that CTG repeat expansions or contractions were frequent events in human cells, that could be evaluated over intervals Drug_discovery as short as 4�C8 weeks, provided that the CTG repeat was highly expanded and actively transcribed.13 To study ASO effects continuously over several weeks we took advantage of the capacity of HT1080 human fibrosarcoma cells for free uptake of oligonucleotides from the culture media.