TEMPLATE-DIRECTED PHOTOLIGATION OF OLIGODEOXYRIBONUCLEOTIDES VIA 4-THIOTHYMIDINE

Non-enzymatic, template-directed ligation of oligonucleotides in aqueo us solution has been of great interest because of its potential synthe tic and biomedical utility and implications for the origin of life. Th ough there are many methods for template-directed chemical ligation of oligonucleotides, there are only three reported photochemical methods . In the first report, template-directed photoligation was effected by cyclobutane dimer formation between the 5'- and 3'-terminal thymidine s of two oligonucleotides with >290 nm light, which also damages DNA i tself. To make the photochemistry of native DNA more selective, we hav e replaced the thymidine at the 5'-end of one oligonucleotide with 4-t hiothymidine (s(4)T) and show that it photoreacts at 366 nm with a T a t the 3'-end of another oligonucleotide in the presence of a complemen tary template, When a single mismatch is introduced opposite either th e s4T or its adjoining T, the ligation efficiency drops by a factor of five or more. We also show that by linking the two ends of the oligon ucleotides together, photoligation can be used to form circular DNA mo lecules and to 'photopadlock' circular DNA templates. Thus, s(4)T-medi ated photoligation may have applications to phototriggered antisense-b ased or antigene-based genetic tools, diagnostic agents and drugs, esp ecially for those situations in which chemical or enzyme-mediated liga tion is undesirable or impossible, for example inside a cell.