Nucleoside 5-triphosphates are the building blocks to synthesis of nucleic acids. Nucleic acids (RNA and DNA) participate in many important biological functions in living systems, including genetic information storage, gene expression, and catalysis. Nucleoside 5- triphosphates have many important therapeutic and diagnostic applications. To understand how these triphosphates are utilized in living systems, numerous synthetic mimics have been prepared and used as active metabolites of certain drugs and molecular probes. Over the years, nucleic acids have been modified at the nucleobase, sugar moiety and phosphate backbone with the aim of understanding their structures and functions. We have site-specifically replaced selected oxygen atoms of nucleosides and nucleotides with selenium atom in order to enzymatically synthesize selenium-derivatized DNAs for obtaining insights into the DNA flexibility, duplex recognition and stability. Although triphosphates have important biological and medicinal significance, they are however, very difficult to synthesize and isolate in high purity and yield. There are many approaches to the synthesis of nucleoside 5-triphosphates, but there is no general strategy that allows simple and direct synthesis of nucleoside triphosphates. To face the challenges, we have developed a new approach in the absence of protecting groups to quickly and efficiently synthesized native deoxynucleoside 5-triphosphates and deoxynucleoside 5-(α- P-seleno)- P-seleno)triphosphates. Syntheses of the triphosphates containing selenium-derivatized nucleobases were also successfully accomplished. After replacing the oxygen atoms at the 4-position of thymidine and uridine, and the 6-position of guanosine, we observed most strikingly, a large bathrochromic shift of over 100 nm, relative to their native counterparts of UV absorbance of 260 nm. Consequently, the synthesized selenium base modified triphosphates are yellow. We also synthesized 2-selenothymidine and 5-methylseleno thymidine 5-triphosphates. We conducted stability study on the colored 4-selenothymidine and used the 5- triphosphate analog (4-SeTTP) as substrate for polymerase recognition. The Klenow polymerase incorporated the 4-SeTTP with efficiency equal to that of the native counterpart. Finally, 4-SeTTP was used to demonstrate UVdamage resistance of selenium-derivatized DNAs and plasmid.