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.
Pongracz
K
K
Department of Neurological Surgery, University of California San Francisco, School of Medicine 94143-0806.
Dosanjh
M K
MK
Singer
B
B
Bodell
W J
WJ
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