Probing the structure of multi-stranded guanine-rich DNA complexes by Raman spectroscopy and enzymatic degradation.
The multi-stranded DNA complexes formed by the oligonucleotides d(T15G4T2G4), Tel, and d(T15G15), TG, were examined by nuclease digestion and Raman spectroscopy. Both Tel and TG can aggregate to form structures consisting of multiple, parallel-oriented DNA strands with two independent structural domains. Overall, the structures of the TG and Tel aggregates appear similar. According to the Raman data, the majority of bases are in C2'-endo/anti conformation. The interaction of guanines at the 3'-ends in both complexes stabilizes the complexes and protects them from degradation by exonuclease III. The 5'-extensions remain single-stranded and the thymines are accessible to single-strand-specific nuclease digestion. The extent of enzymatic cleavage at the junction at the 5' end of the 15 thymines implies a conformational change between this part of the molecule and the guanine-rich region. The differential enzymatic sensitivity of the complexes suggests there are variations in backbone conformations between TG and Tel aggregates. TG aggregates were more resistant to digestion by DNase I, Mung Bean nuclease, and S1 nuclease than Tel complexes. It is proposed that the lower DNase I sensitivity may be partly due to the more stable backbone exhibited by TG than Tel complexes. Structural uniformity along the guanine core of TG is suggested, as there is no indication of structural discontinuities or protected sites in the guanine-rich regions of TG aggregates. The lower extent of digestion by Mung Bean nuclease at the 3' end implies that these bases are inaccessible to the enzyme. This suggests that there is minimal fraying at the ends, which is consistent with the extreme thermal stability of the TG aggregates.
Poon
K
K
Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Ontario, Canada.
Macgregor
R B
RB
D001482:Base Composition; D004247:DNA / Q000737:chemistry*; D003850:Deoxyribonuclease I; D004591:Electrophoresis, Polyacrylamide Gel; D005090:Exodeoxyribonucleases; D006147:Guanine / Q000737:chemistry; D066298:In Vitro Techniques; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013059:Spectrum Analysis, Raman
D009838:Oligodeoxyribonucleotides; D006147:Guanine; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D003850:Deoxyribonuclease I; D015719:Single-Strand Specific DNA and RNA Endonucleases