Many neurological disorders are caused by expanded trinucleotide repeats1, including Machado-Joseph Disease (MJD)2 and Huntington Disease (HD)3. MJD and HD are caused by expanded CAG repeats within the ataxin-3 (ATXN3) and huntingtin (HTT) genes. Inhibiting expression of ATXN3 or HTT are promising therapeutic strategies, but indiscriminant inhibition of wild-type and mutant alleles may lead to toxicity. We hypothesized that expanded triplet repeat mRNA might be preferentially recognized by complementary oligomers. We observe selective inhibition of mutant ataxin-3 and HTT protein expression by peptide nucleic acid (PNA) and locked nucleic acid (LNA) oligomers targeting CAG repeats. Duplex RNAs were less selective, suggesting an advantage for single-stranded oligomers. Inhibiting mutant HTT expression protected cultured striatal neurons from an HD mouse model against glutamate-induced toxicity. Antisense oligomers that discriminate between wild-type and mutant genes on the basis of repeat length offer new options for treating MJD, HD, and other hereditary diseases.

Oligonucleotides containing locked nucleic acid bases (LNAs) have increased affinity for complementary DNA sequences. We hypothesized that enhanced affinity might allow LNAs to recognize chromosomal DNA inside human cells and inhibit gene expression. To test this hypothesis, we synthesized antigene LNAs (agLNAs) complementary to sequences within the promoters of progesterone receptor (PR) and androgen receptor (AR). We observed inhibition of AR and PR expression by agLNAs but not by analogous oligomers containing 2'-methoxyethyl bases or noncomplementary LNAs. Inhibition was dose dependent and exhibited IC 50 values of <10 nM. Efficient inhibition depended on the length of the agLNA, the location of LNA bases, the number of LNA substitutions, and the location of the target sequence within the targeted promoter. LNAs targeting sequences at or near transcription start sites yielded better inhibition than LNAs targeting transcription factor binding sites or an inverted repeat. These results demonstrate that agLNAs can recognize chromosomal target sequences and efficiently block gene expression. agLNAs could be used, for gene silencing, as cellular probes for chromosome structure, and therapeutic applications.The development of oligonucleotides and oligonucleotide mimics for sequence-specific recognition of chromosomal DNA inside cells confronts several challenges (1). Compounds must be able to enter cells, pass into the nucleus, and bind chromosomal DNA with high specificity. Binding must occur in spite of base-pairing at the target site and complexation of the genomic sequence with histones, transcription factors, and other DNA binding proteins. Once bound, the association of an oligomer with the chromosome must be sufficiently stable and long-lasting to affect gene expression. These challenges make recognition of DNA more complex than recognition of mRNA and overcoming them requires understanding the chemical, biophysical, and biological properties of native nucleic acids and their chemically modified analogs and mimics.We have shown that peptide nucleic acids 1 (PNAs) (Figure 1), a class of DNA/RNA mimic with an uncharged amide backbone (2,3), can target chromosomal DNA at transcription start sites and inhibit gene expression inside cells (4,5). Inhibition of gene expression by promotertargeted antigene PNAs (agPNAs, we use the term antigene to differentiate molecules that are complementary to chromosomal DNA from antisense oligomers that are complementary to mRNA) demonstrated that synthetic molecules could access sequence information at transcription start sites and that binding was sufficient to block expression.To whom correspondence should be addressed. Email: david.corey@utsouthwestern The PNAs used in our previous studies were mixed sequences (i.e. containing all four PNA bases) and were designed to recognize their targets by Watson-Crick base-pairing and strand invasion rather than by triple helix formation (4,5). With their neutral amide backbone and propensity to invade duplex DNA, PNAs have u...

Sequence-selective recognition of DNA inside cells by oligonucleotides would provide valuable insights into cellular processes and new leads for therapeutics. Recent work, however, has shown that noncoding RNA transcripts overlap chromosomal DNA. These RNAs provide alternate targets for oligonucleotides designed to bind promoter DNA, potentially overturning previous assumptions about mechanism. Here, we show that antigene locked nucleic acids (agLNAs) reduce RNA levels of targeted genes, block RNA polymerase and transcription factor association at gene promoters, and bind to chromosomal DNA. These data suggest that the mechanism of LNAs involves recognition of chromosomal DNA and that LNAs are bona fide antigene molecules.Chromosomal DNA is an important target for molecular recognition by synthetic agents (1). Research over thirty years has examined triple-helix forming agents (2), oligomers that bind through Watson-crick base pairing (3), and small molecule pyrimidine polyamides (4). These compounds share a common assumption-that recognition would occur through binding to DNA.Recently, double stranded antigene RNA (agRNAs) (we use the term "antigene" to distinguish these short RNAs from similar duplexes that are complementary to mRNA) that target transcription start sites for gene promoters have been shown to modulate gene expression (5-8). Expression increases in a cell lines where endogenous expression is relatively low, and decreases in a cell line where endogenous expression is relatively high.We had initially hypothesized that agRNAs were directly interacting with chromosomal DNA inside cells. However, we discovered that the interaction was mediated by the protein argonaute 2 (AGO2) (6). AGO2 is known to promote formation of RNA-RNA hybrids (9), rather than DNA-RNA hybrids, bringing the likelihood of DNA-RNA binding into question.We then investigated the molecular target for agRNAs (8). We discovered that DNA was not the only potential target, there was also a noncoding RNA transcript overlapping the promoter that originated within the target gene and was transcribed in an antisense orientation. We showed that agRNAs were binding to this transcript, recruited AGO protein to the transcript, and that reduced expression of the transcript reversed the action of activating agRNAs. Together, these data suggested that the noncoding RNA, not chromosomal DNA, was the direct target for agRNAs. † This work was supported by the National Institutes of Health (NIGMS 60642 and NIGMS 73042) The presence of noncoding RNA transcripts that overlap genes is a widespread phenomenon. Over 20% of genes have antisense transcripts and over 80% of the genome is transcribed into RNA (11). The purpose of these transcripts is a major unresolved question for biology. What is certain is that these transcripts complicate previously easy assumptions about the mechanism of action of antigene agents originally designed to target chromosomal DNA.LNA nucleotides contain a methylene bridge connecting the 2′-oxygen of the ribose with ...