NMR of enzymatically synthesized uniformly 13C15N-labeled DNA oligonucleotides.
AUTOR(ES)
Zimmer, D P
RESUMO
A procedure for the enzymatic synthesis of uniformly 13C15N-labeled DNA oligonucleotides in milligram quantities for NMR studies is described. Deoxynucleotides obtained from microorganisms grown on 13C and 15N nutrient sources are enzymatically phosphorylated to dNTPs, and the dNTPs are incorporated into oligonucleotides using a 3'-5' exonuclease-deficient mutant of Klenow fragment of DNA polymerase I and an oligonucleotide template primer designed for efficient separation of labeled product DNA from unlabeled template. The labeling strategy has been used to uniformly label one or the other oligonucleotide strand in the DNA duplex dGGCAAAACGG.dCCGTTTTGCC in order to facilitate assignment and structure determination by NMR. Application of 15N and 13C heteronuclear NMR experiments to isotopically labeled DNA is presented.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=42110Documentos Relacionados
- Simple, efficient protocol for enzymatic synthesis of uniformly 13C, 15N-labeled DNA for heteronuclear NMR studies.
- Preparation and characterization of a uniformly 2 H/ 15 N-labeled RNA oligonucleotide for NMR studies.
- Complete resolution of the solid-state NMR spectrum of a uniformly 15N-labeled membrane protein in phospholipid bilayers
- 15N NMR study of a mixture of uniformly labeled tRNAs.
- Rapid non-radioactive TMACl hybridization protocol employing enzymatically labeled oligonucleotides.
