Anthony J. Shallop

Modulation of RNA Metal Binding by Flanking Bases: 15N NMR Evaluation of GC, Tandem GU, and Tandem GA Sites

15N NMR chemical shift changes in the presence of Mg(H2O)6 2+, Zn2+, Cd2+, and Co(NH3)6 3+ were used to probe the effect of flanking bases on metal binding sites in three different RNA motifs. We found that: for GC pairs, the presence of a flanking purine creates a site for the soft metals Zn2+ and Cd2+ only; a GG·UU motif selectively binds only Co(NH3)6 3+, while a UG·GU motif binds none of these metals; a 3′ guanosine flanking the adenosine of a sheared GA·AG pair creates an unusually strong binding site that precludes binding to the cross-strand stacked guanosines within the tandem pair.

Use of 13C tags with specifically 15N-labeled DNA and RNA

We describe methods to introduce 13C specifically to the C2 or C8 positions of 15N‐multilabeled purines, thereby permitting “13C tagging” of all base nitrogens except the amino group of adenosine. These procedures permit the incorporation of two or more 15N‐multilabeled monomers into a given DNA or RNA fragment, with and without appropriate 13C tags. This approach increases the number of 15N‐nmr resonances that can be unambiguously distinguished and thus the amount of information from a single synthesis and a single nmr experiment.

Use of both direct and indirect 13C tags for probing nitrogen interactions in hairpin ribozyme models by 15N NMR.

We have used the synthesis and 15N NMR study of separate loop A and loop B domains of the hairpin ribozyme to demonstrate that multiple 15N atoms can be incorporated into an RNA strand and be unambiguously distinguished through a combination of direct and indirect tagging by 13C atoms. Absence of 15N chemical shift changes shows that the G8N1 in loop A does not become deprotonated up to pH 8, and that the G21N7 of loop B does not bind to Mg2 +. †In honor and celebration of the 70th birthday of Professor Leroy B. Townsend.