A new photoactive building block for investigation of DNA backbone interactions: photoaffinity labeling of human DNA polymerase beta.
The cross-linking of target proteins or nucleic acids to light-activatable ligands is an important tool for elucidating molecular interactions. Through the use of photoaffinity-labeling reagents, several new insights into nucleic acid interactions have been obtained, for example in DNA replication and repair. In most known photoprobes, the applied light-sensitive functionalities are placed directly at the nucleobase or are attached via linkers to either the nucleobase or the phosphate backbone. Here we describe the first photoprobe that bears a light-sensitive aryl(trifluoromethyl)diazirine at the sugar moiety of a DNA oligonucleotide. We devised a route for the synthesis of the modified nucleoside and its incorporation into an oligonucleotide. The photoactive species was proven to be stable under the conditions employed in routine automated DNA synthesis. The modified oligonucleotide was shown by subsequent photolabeling studies of human DNA polymerase beta to form a covalent complex to the enzyme upon irradiation with near-UV light.
Liebmann
Meike
M
Fachbereich Chemie, Universität Konstanz, Universitätsstrasse 10, 78457 Konstanz, Germany.
Di Pasquale
Francesca
F
Marx
Andreas
A
D004247:DNA / Q000737:chemistry*; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000528:radiation effects; D006801:Humans; D015394:Molecular Structure; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000528:radiation effects; D019920:Photoaffinity Labels / Q000737:chemistry* / Q000528:radiation effects; D011485:Protein Binding; D013936:Thymidine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000528:radiation effects; D014466:Ultraviolet Rays
D009841:Oligonucleotides; D019920:Photoaffinity Labels; D004247:DNA; D019951:DNA Polymerase beta; D013936:Thymidine