Thermodynamic and Structural Studies of Single Nucleotide Bulges in Nucleic Acids.

Darryl Andre Leblanc, Louisiana State University and Agricultural & Mechanical College


The single nucleotide bulge is a nucleic acid secondary structural motif of important biological relevance. Bulges in DNA function as intermediates in the process of frameshift mutation. Bulges in RNA participate in protein-RNA interactions, RNA tertiary interactions, and ribozyme functioning. UV-absorbance methods were used to obtain the thermodynamic parameters for the helix-to-coil transition for the deoxyribooligonucleotides shown below.(UNFORMATTED TABLE OR EQUATION FOLLOWS)$$\vbox{\halign{#\hfil&&\quad#\hfil\cr&dGCGAAXAAGCG& dGCGAA-AAGCG&dGCGAAAAGCG\cr&dCGCTT-TTCGC&dCGCTTZTTCGC&dCGCTTTTCGC\cr &X = G,C or T&Z = G,C or A&Decamer\cr}}$$(TABLE/EQUATION ENDS)All of the duplexes with bulges are destabilized relative to the Decamer by 3.5-4.5 kcal/mol at 37$\sp\circ$C. The identity of the bulge and its sequence context were found to have small effects on the relative stabilities of the molecules that contain bulges. Purine bulges were found to be slightly more destabilizing than pyrimidine bulges. Bulges located between two pyrimidines were found to be slightly more destabilizing than those located between two purines. UV-absorbance methods were utilized to examine the thermodynamics and mechanism of ethidium binding to the decamer and the duplexes above where X = Z = G. Ethidium intercalates into the decamer with a small preference for the sequence 5$\sp\prime$CpG3$\sp\prime$. For Z = G above, results were consistent with previous studies indicating two strong binding sites adjacent to the bulge (Nelson & Tinoco, Jr., 1985). However, several models fit the data well. The results for X = G above were anomalous. Multidimensional $\sp1$H, $\sp{13}$C and $\sp{15}$N nuclear magnetic resonance were used to study the structure of a single adenine bulge located in the stem of an RNA hairpin 25 nucleotides in length. The bulge and surrounding sequence are highly conserved among 16S ribosomal RNAs. NOESY data indicate an intrahelical conformation for the bulge. Estimation of ribose H1$\sp\prime$-H2$\sp\prime$ coupling constants and supporting NOESY data indicate that the bulged adenine and the 3$\sp\prime$ neighboring cytosine adopt the C2$\sp\prime$-endo or DNA-like conformation, rather than the C3$\sp\prime$-endo conformation typical of A-form RNA helices. These results are discussed in the context of a preliminary molecular model.