Biomedical subjects

James L Hougland

Publications and source records attributed to James L Hougland.

3 recordsLinked to original sources

PubMed2005

Improved synthesis of 2'-amino-2'-deoxyguanosine and its phosphoramidite.

2'-Amino-2'-deoxynucleosides and oligonucleotides containing them have proven highly effective for an array of biochemical applications. The guanosine analogue and its phosphoramidite derivatives have been accessed previously from 2'-amino-2'-deoxyuridine by transglycosylation, but with limited overall efficiency and convenience. Using simple modifications of known reaction types, we have developed useful protocols to obtain 2'-amino-2'-deoxyguanosine and two of its phosphoramidite derivatives with greater convenience, fewer steps, and higher yields than reported previously. These phosphoramidites provide effective synthons for the incorporation of 2'-amino-2'-deoxyguanosine into oligonucleotides.

Deoxyguanosine↗

PubMed2005

Functional identification of catalytic metal ion binding sites within RNA.

The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (MA, MB, and MC) that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-SP phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for MC. Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis.

Animals↗

PubMed2004

An atomic mutation cycle for exploring RNA's 2'-hydroxyl group.

The 2'-hydroxyl group fulfills numerous structural and functional roles in RNA, including those of hydrogen bond donor and acceptor. While loss of function upon 2'-deoxynucleotide substitution establishes the importance of specific 2'-hydroxyl groups within RNA, this approach provides no information about how these hydroxyl groups impart their functional contribution. We use an atomic mutation cycle to evaluate the functional importance of the 2'-hydroxyl group's hydrogen atom. Using the Tetrahymena ribozyme reaction, we challenge the cycle to expose the catalytic contribution of the cleavage site 2'-hydroxyl group and its associated hydrogen bond network. The results establish the viability of this cycle as an approach to reveal 2'-hydroxyl groups that donate functionally significant hydrogen bonds.

Hydrogen Bonding↗