Synthesis of a 25 base oligonucleotide containing a styrene oxide modification at the O6 position of 2'-deoxyguanosine at a defined site and incorporation studies of the similarly modified 2'-deoxyguanosine-5'-triphosphate.
A diastereomeric mixture of the regioisomers O6-(2-hydroxy-2-phenylethyl)-2'-deoxyguanosine (st6G, beta-isomer) and O6-(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine (alpha-isomer) was site-specifically placed in a 25 base oligonucleotide template 5'-CCGCTAst6GCGGGTACCGAGCTCGAAT-3' using CED phosphoramidite chemistry. Using 32P-post-labeling we found the oligonucleotide to contain 95% of the beta-isomer and 5% of the alpha-isomer of st6G. st6G as the 3'-phosphate was found to be considerably more acid labile than O6-methyl-2'-deoxyguanosine-3'-phosphate, leading to dealkylation during oligonucleotide synthesis. The diastereomeric mixture of O6-(2-hydroxy-2-phenylethyl)-2'-deoxy-guanosine-5'-triphosphate (st6dGTP) was chemically synthesized and used as a substrate for the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I. This study demonstrated that st6dGTP could be incorporated opposite deoxycytidine and did not completely block replication.
Department of Neurological Surgery, University of California San Francisco, School of Medicine 94143-0806.
K
K
Pongracz
M K
MK
Dosanjh
B
B
Singer
W J
WJ
Bodell
D001483:Base Sequence; D003848:Deoxyguanine Nucleotides / Q000737:chemistry*; D003849:Deoxyguanosine / Q000737:chemistry*; D004852:Epoxy Compounds / Q000737:chemistry*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000138:chemical synthesis*; D013237:Stereoisomerism; D013698:Templates, Genetic
D003848:Deoxyguanine Nucleotides; D004852:Epoxy Compounds; D009841:Oligonucleotides; C029603:deoxyguanosine triphosphate; C013690:styrene oxide; D003849:Deoxyguanosine