Syntheses and Nitrogen-15 NMR of specifically labeled DNA fragments for assignment and RNA fragments to probe metal binding

High yield procedures for protection of purine ribonucleosides for phosphoramidite RNA synthesis have been developed. A new reaction which allows concomitant highly regiospecific 2'-silylation and 3' -phosphitylation gives fully protected 3'(2 ')-H-phosphonates with excellent selectivity (85--90:10--15). Subsequent dephosphonylation of the H-phosphonate monoester moiety without silyl migration using polyhydroxy alcohols provides intermediates ready for phosphitylation by standard methods to give fully protected phosphoramidites.; Similar dephosphonylations with polyhydroxy alcohols have been applied to the problem of recycling excess phosphoramidites. Hydrolysis of an activated nucleoside phosphoramidite to a 3'-cyanoethyl-H-phosphonate diester followed by a facile transesterification with ethylene glycol or glycerol results in a two-step dephosphonylation. The mild conditions allow this approach to be applied to recycling ribonucleoside phosphoramidites having a 2 '-O-tertbutyldimethylsilyl (BDMS) protecting groups without isomerization. Alternatively, reaction of an activated nucleoside phosphoramidite with ethylene glycol or glycerol directly, without prior hydrolysis to the H-phosphonate diester, leads to dephosphonylation in a one-flask procedure.; Specifically 15N-labeled DNA oligonucleotides have been synthesized in sufficient quantities (1--5 mumol) for NMR by the H-phosphonate method. Several labeled DNA 15mers were used in the unambiguous assignments of the imino and amino protons in an L-argininamide-DNA aptamer complex. These assignments are crucial for the successful determination of the solution structure of this complex. 15N NMR of a labeled DNA duplex containing a GCN4 half binding site implicates binding between a modified GCN4 peptide and the DNA.; Specifically 15N-labeled RNA oligonucleotides have been synthesized in ample quantities (up to 20 mumol) by the phosphoramidite method. 15N NMR of these RNA fragments shows site-specific binding of Mg2+ and Zn2+ to several small conserved RNA motifs, including a GC cluster, cross-strand purine stacks, and an E-element. These results demonstrate that 15N NMR of specifically labeled RNA fragments is a direct and sensitive way to probe and evaluate RNA-metal binding sites.