X-ray studies on triple-stranded nucleic acids

Andrea Giacometti, Purdue University

Abstract

Synthetic duplexes of homopolymer DNAs like poly d(T)$\cdot$poly d(A) and RNAs like poly (U)$\cdot$poly (A) and poly (I)$\cdot$poly (A) can combine with another polynucleotide chain to form triple-stranded complexes, poly (dT)$\cdot$poly (dA)$\cdot$poly (dT), poly (U)$\cdot$poly (A)$\cdot$poly (U) and poly (I)$\cdot$poly (A)$\cdot$poly (I) respectively, in which the additional chain is Hoogsteen-base-paired to the middle polypurine nucleotide chain and runs parallel to it. The available x-ray diffraction patterns from oriented fibers of these three triple-stranded nucleic acids have been digitized with an Optronics film scanner, the raw intensity data have been processed using a Lexidata system and have been analyzed by Linked-Atom Least-Squares procedure, and the final detailed structures are presented in this thesis. Eight 12-fold helical models of pitch 38.4 A have been generated for poly d(T)$\cdot$poly d(A)$\cdot$poly d(T) incorporating C3$\sp\prime$-endo or C2$\sp\prime$-endo sugar puckering for each of the three strands and these models have been refined against contacts and X-ray data. The best model corresponds to C2$\sp\prime$-endo sugar pucker in all three strands and has an R = 0.29. Eight 11-fold helical models of pitch 33.5 A have been produced for poly (U)$\cdot$poly (A)$\cdot$poly (U) corresponding to C2$\sp\prime$-endo or C3$\sp\prime$-endo sugar puckering for each of the three strands. These models have been tested for contacts and refined against the X-ray data. The only one that survived the tests incorporates C3$\sp\prime$-endo for the uracil strand of the Watson-Crick base pair and C2$\sp\prime$-endo for the others. The R is 0.25. That indicates a very good agreement between the X-ray intensities calculated from the model and those observed in the diffraction pattern. Eight 12-fold helical models of pitch 39.7 A for poly (I)$\cdot$poly (A)$\cdot$poly (I), incorporating C2$\sp\prime$-endo or C3$\sp\prime$-endo sugar puckering for each of the three strands, have been produced and refined against the observed X-ray intensities. The model corresponding to all C3$\sp\prime$-endo sugar puckering has shown to be the best with an R = 0.24. The structural features emerging from this study on molecular diameter, groove dimensions, base stacking interactions and interchain hydrogen bonds provide sound explanation for the higher stability of triple-stranded nucleic acids than those observed for the corresponding duplexes alone.

Degree

Ph.D.

Subject Area

Molecular biology|Genetics

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