Solid-phase synthesis and high-resolution NMR studies of two synthetic double-helical RNA dodecamers: r(CGCGAAUUCGCG) and r(CGCGUAUACGCG).
Ten-micromole solid-phase RNA synthesis has been successfully performed on an automated nucleic acid synthesizer with coupling efficiencies up to 99%, using the tert-butyldimethylsilyl group to protect the 2'-hydroxyl. The tert-butyldimethylsilyl group was easily removed by tetrabutylammonium fluoride under conditions in which virtually no 2'- to 3'-isomerization was found to occur. By use of this approach, the self-complementary RNA dodecamers r(CGCGAAUUCGCG) and r(CGCGUAUACGCG) were synthesized on an automated nucleic acid synthesizer, purified by TLC, and studied by high-resolution NMR. Imino protons were assigned from one-dimensional nuclear Overhauser effects. The nonexchangeable base, H1', and H2' protons were assigned by the sequential NOESY connectivity method. The NOE data from these two oligomers were analyzed qualitatively and compared to the ideal A- and B-type helix models of Arnott et al. (1972a,b). The internucleotide H6/H8 NOEs to the preceding H1' in r(CGCGUAUACGCG) were found to be sequence-dependent and probably reflect the roll angles between adjacent bases. The internucleotide H6/H8 to H2' NOEs of these oligomers correspond very well to an A-type conformation, but the interstrand adenine H2 NOEs to the following H1' were much stronger than those predicted from the fiber model. These srong interstrand NOEs can be rationalized by base pair slide to favor more interstrand base overlap, as predicted by Callidine and Drew (1984).
Chou
S H
SH
Howard Hughes Medical Institute, Biochemistry Department, University of Washington, Seattle 98195.
Flynn
P
P
Reid
B
B
D001482:Base Composition; D003196:Computer Graphics; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000138:chemical synthesis*; D012313:RNA / Q000138:chemical synthesis*; D012997:Solvents
D007202:Indicators and Reagents; D009843:Oligoribonucleotides; D012997:Solvents; D012313:RNA