Template Independent Synthesis of Nucleic Acid Libraries - Template Independent Synthesis of Nucleic Acid Libraries - Open Access Pub

Though, directed evolution/In vitro evolution has greatly enhanced the applicability of natural biomolecules, there is still a big void in synthetic biology, which could be filled only when we are able to make novel/synthetic functional biomolecules. Terminal deoxyribonucleotidyl transferase (TdT) is the only known DNA polymerase, which can add deoxyribonucleotides without the requirement of a DNA template. Here, we are introducing the concept of Template-Independent Synthesis of Nucleic Acids (TISNA), where we have exploited the property of terminal deoxyribonucleotidyl transferase to add deoxyribonucleotides to the 3’ end of an oligonucleotide for the generation of de novo libraries of ssDNA, dsDNA coding sequences and RNA. We are able to generate libraries that have diversity not only in sequence but also in length in a single library itself. The length of double stranded random gene libraries generated using this approach ranges from 200 base pairs to 10 kilobase pairs. The ability to make random nucleic acid libraries from scratch (independent of any template information) in the laboratory could open up new avenues and holds promise for the pharmaceutical and biotechnological sectors. DOI10.14302/issn.2575-7881.jdrr-17-1749 There is no denying the fact that nucleic acids and proteins made after billion years of natural evolution are serving the host organism in the best way possible; still the applicability of the existing biomolecules for humans still has a lot to be desired 1. The first land mark work in the field of synthetic biology was the synthesis of the first complete gene, a yeast tRNA, as demonstrated by HarGobind Khorana 2. Few years ahead, synthesis of the first peptide- and protein-coding genes was done in the laboratories of Herbert Boyer and Alexander Markham, respectively 3, 4. Szostak reported for the first time that genuinely new enzymatic activities could be created de novo without the need for prior mechanistic information by selection from a naive protein library of very high diversity 5, 6. The chemically synthesized library which they had generated was limited by the size as the length of randomized nucleotide addition was restricted only to 200-300 bases. To generate higher sized sequences, different parts (more than 150 bases) were made separately and later on attempted for ligation, but the overall yield was still marginal 7. The biggest challenge in building novel nucleic acids de novo is the limitation of solid-phase DNA synthesis, which does not have the capacity to randomize beyond a couple of hundred bases 8. We