Earlier, we calculated the stereochemically feasible DNA triple helix (the so called R-form, or recombination triplex). A DNA-RNA structure, similar to R-form, was recently detected in the T7 transcription complex studied by iodine-125 radioprobing. The three-stranded associate DNA-DNA-RNA was observed at the """"""""upstream"""""""" end of the transcription """"""""bubble"""""""" (outside the polymerase """"""""cleft""""""""). The functional role of this structure is likely related to stabilization of the elongation transcription complex. It is also possible that this DNA/RNA association is used by the repair enzymes for recognition of mismatches in the DNA duplex. We suggest that the equilibrium between the two states, RNA:DNA+DNA and RNA+DNA:DNA, would be different for the WC pair and a mismatch (in the latter case the equilibrium will be shifted toward RNA:DNA+DNA, and the third DNA strand will be predominantly exposed). To test this hypothesis, we have initiated a new project. Specifically, the three stranded DNA/RNA structures in solution and in transcription elongation complexes are to be analyzed with several spectroscopic methods. To facilitate formation of the intranucleotide R-triplexes and to avoid aggregation, we designed the oligonucleotide sequences based on our calculations. First, only the DNA constructs will be used, and later RNA will be added as well. The equilibrium between the """"""""closed"""""""" and the """"""""open"""""""" states will be monitored by UV, CD and fluorescence measurements. To this aim, the fluorescent pteridine-based nucleoside analog, 6-methylisoxanthopterin (F), will be used as a probe. Our data indicate that the most stable triplexes would have the F-probe at the 5'-end followed by thymine. Importantly, this observation is consistent with the R-form structure calculated by us earlier, and can be explained by means of simple stereochemical considerations. The optimal oligonucleotides are to be studied by NMR. In a second stage, we intend to study the DNA-DNA-RNA triplexes with various mismatches introduced in the DNA strands, including fluorescence measurements in real time. These studies would provide a unique knowledge of the thermodynamics and kinetics of the opening-closing transitions in DNA and RNA during transcription. In addition, these results would be useful for understanding practical aspects, such as the stereochemical mechanisms likely to be involved in the transcription-related recognition of mismatches. Z01 BC 10046-05
