Analysis of the yeast genome: identification of new non-coding and small ORF-containing RNAs.

The genome sequences from increasing numbers of organisms allow for rapid and organized examination of gene expression. Yet current computational-based paradigms for gene recognition are limited and likely to miss genes expressing non-coding RNAs or mRNAs with small open reading frames (ORFs). We have utilized two strategies to determine if there are additional transcripts in the yeast Saccharomyces cerevisiae that were not identified in previous analyses of the genome. In one approach, we identified strong consensus polymerase III promoters based on sequence, and determined experimentally if these promoters drive the expression of an RNA polymerase III transcript. This approach led to the identification of a new, non-essential 170 nt non-coding RNA. An alternative strategy analyzed RNA expression from large sequence gaps>2 kb between predicted ORFs. Fifteen unique RNA transcripts ranging in size from 161 to 1200 nt were identified from a total of 59 sequence gaps. Several of these RNAs contain unusually small potential ORFs, while one is clearly non-coding and appears to be a small nucleolar RNA. These results suggest that there are likely to be additional previously unidentified non-coding RNAs in yeast, and that new paradigms for gene recognition will be required to identify all expressed genes from an organism.

Binding of DNA oligonucleotides to sequences in the promoter of the human bc1-2 gene.

Duplex DNA recognition by oligonucleotide-directed triple helix formation is being explored as a highly specific approach to artificial gene repression. We have identified two potential triplex target sequences in the promoter of the human bcl-2 gene, whose product inhibits apoptosis. Oligonucleotides designed to bind these target sequences were tested for their binding affinities and specificities under pseudo-physiological conditions. Electrophoretic mobility shift and dimethyl sulfate footprinting assays demonstrated that an oligonucleotide designed for simultaneous recognition of homopurine domains on alternate duplex DNA strands had the highest affinity of any oligonucleotide tested. Modifications to render this oligonucleotide nuclease-resistant did not reduce its binding affinity or specificity. In additional studies under various pH conditions, pyrimidine motif complexes at these target sequences were found to be stable at pH 8.0, despite the presumed requirement for protonation of oligonucleotide cytidines. In contrast, purine motif complexes, typically considered to be pH independent, were highly destabilized at decreasing pH values. These results indicate that a natural sequence in the human bcl-2 promoter can form a stable triplex with a synthetic oligonucleotide under pseudo-physiological conditions, and suggest that triple helix formation might provide an approach to the artificial repression of bcl-2 transcription.

Overcoming potassium-mediated triplex inhibition.

Sequence-specific duplex DNA recognition by oligonucleotide-directed triple helix formation is a possible approach to in vivo gene inhibition. However, triple helix formation involving guanine-rich oligonucleotides is inhibited by physiological ions, particularly K+, most likely due to oligonucleotide aggregation via guanine quartets. Three oligodeoxynucleotide (ODN) derivatives were tested for their ability to resist guanine quartet-mediated aggregation, yet form stable triplexes. Electrophoretic mobility shift and dimethyl sulfate footprinting assays were used to analyze the formation of triplexes involving these oligonucleotide derivatives. In the absence of K+, all ODNs had similar binding affinities for the duplex target. Triplexes involving a 14mer ODN derivative containing 7-deazaxanthine substituted for three thymine bases or an 18mer ODN containing two additional thymines on both the 5' and 3' termini were abolished by 50 mM K+. Remarkably, triplexes involving an ODN derivative containing four 6-thioguanine bases substituted for guanine resisted K+ inhibition up to 200 mM. We hypothesize that the increased radius and decreased electronegativity of sulfur in the 6-position of guanine destabilize potential guanine quartets. These results improve the prospects for creating ODNS that might serve as specific and efficient gene repressors in vivo.

Competitive triplex/quadruplex equilibria involving guanine-rich oligonucleotides.

Oligonucleotide-directed triple helix formation in the purine motif involves the binding of guanine-rich oligonucleotides to duplex DNA. Although this approach has been proposed for in vivo gene inhibition, triple helix formation by guanine-rich oligonucleotides is severely inhibited by physiological concentrations of certain monovalent cations (M+), especially K+. To clarify the mechanism of this inhibition, electrophoretic gel mobility shift titrations were performed to analyze the formation and stability of a purine motif triple helix in the presence of M+ and to monitor oligonucleotide aggregation under these conditions. M+ inhibition of triplex formation exhibited a concentration and ionic radius dependence that correlates with the ability of M+ to stabilize guanine quartet structures. In the presence of inhibitory [M+], guanine-rich oligonucleotides formed aggregates having characteristics consistent with the involvement of guanine quartets. The inhibitory effects of K+ on triplex formation could not be reversed by addition of the physiological polyamines spermidine3+ or spermine4+. M+ reduced the equilibrium concentration of the triplex primarily by decreasing the rate of triplex formation, but M+ also caused a detectable increase in the rate of triplex dissociation. Together, these results suggest that triplex inhibition under physiological ionic conditions is caused by competing equilibria wherein guanine-rich oligonucleotides form aggregates involving guanine quartets. Approaches to destabilizing aggregates of guanine-rich oligonucleotides under physiological conditions will be required before in vivo applications can be realistically considered.

DNA recognition by alternate strand triple helix formation: affinities of oligonucleotides for a site in the human p53 gene.

Duplex DNA recognition by oligonucleotide-directed triple helix formation is generally limited to homopurine target domains. Various approaches have been suggested for the relief of this constraint. Artificial DNA sequences have previously been used to show that adjacent homopurine domains on opposite DNA strands can be simultaneously recognized by oligonucleotide probes that switch triple helix recognition motifs between domains. Using assays of electrophoretic mobility and chemical protection, we have explored in detail whether such strategies are of benefit in designing high-affinity probes for a natural DNA sequence in the human p53 gene. This target site contains three adjacent, purine-rich domains on opposite DNA strands. Our results show that (i) a modest but statistically significant enhancement in affinity can be achieved for this sequence by designing an oligonucleotide that simultaneously recognizes all three purine domains, (ii) correction of a pyrimidine interruption in one purine domain does not dramatically alter this result, (iii) the relative energetic and structural contributions attributable to recognition of each purine domain can be assessed using probes with combinations of specific and nonspecific nucleotide sequences, and (iv) probe affinity is not correlated with the apparent number of base triplets for certain complexes. These data suggest that unfavorable free energy changes may be associated with alternation between triple helix motifs using existing strategies. In contrast to artificial DNA sequences optimized for this purpose, a substantial affinity enhancement was not observed using alternate strand DNA recognition at this natural target sequence. We therefore conclude that such enhancement is sequence dependent.

Analysis of duplex DNA by triple helix formation: application to detection of a p53 microdeletion.

Conventional methods for mutation detection include Southern hybridization, direct sequencing of PCR products and single-strand conformation polymorphism analysis. We present an additional screening method that employs oligonucleotide-directed DNA triple helix formation to detect mutations within homopurine sequences. The proposed strategy is simple and may be of particular value when screening many DNA samples for changes involving particular homopurine sites. We have applied the method to the analysis of a clinically relevant 8-bp micro-deletion in the human p53 tumor suppressor gene. Affinities of oligonucleotide probes toward radiolabeled wild-type and mutant p53 DNA duplexes were quantitated by electrophoretic mobility shift assays. Recombinant plasmids carrying wild-type or microdeleted forms of the p53 homopurine sites of interest were created. Dimethyl sulfate footprinting was used to verify intended probe specificities. Duplex PCR products amplified from plasmid constructs were directly probed by incubation with labeled oligonucleotides. After electrophoresis and autoradiography, patterns of triple helix formation allowed discrimination between the mutant and wild-type p53 sequences. Direct DNA analysis by triple helix formation may simplify other procedures that normally require DNA denaturation and hybridization.