title: Photo-cleavable nucleotides for primer free enzyme mediated DNA synthesis.
abstract: The synthesis, characterization and potential application of eight 3'-O modified 2'-deoxyribonucleoside triphosphates (dNTPs) are discussed. These nucleotide analogues are modified by capping the 3'-OH with a photolabile protecting group which can temporarily cease DNA strand growth and can smoothly reinitiate the growth by the photodecomposition of the protecting group and setting the 3'-OH of dNTPs free to propagate. The synthesis of 3'-O-(2-nitrobenzyl)-2'-deoxyribonucleoside triphosphates (NB-dNTPs) and 3'-O-(4,5-dimethoxy-2-nitrobenzyl)-2'-deoxyribonucleoside triphosphates (DMNB-dNTPs) is discussed in detail with structural confirmation using NMR. The UV-cleaving studies are monitored and quantified using LCMS and (1)H NMR spectral traces. The synthesised nucleotides are employed for terminating and reinitiating template-less DNA synthesis, using primer independent Terminal Deoxynucleotidyl Transferase (TdT) enzyme. The use of this photolabile nucleotide in one step stop-start DNA synthesis is a novel strategy towards the precise assembly of dNTPs with the potential to reinforce present technologies.
authors.affiliation: Ingenuity Lab, 11421 Saskatchewan Drive, Edmonton, Alberta T6G 3M9, Canada. montemag@ualberta.ca.
authors.forename: Anu Stella
authors.initials: AS
authors.lastname: Mathews
authors.forename: Haikang
authors.initials: H
authors.lastname: Yang
authors.forename: Carlo
authors.initials: C
authors.lastname: Montemagno
mesh_terms: D004247:DNA / Q000737:chemistry*; D053837:DNA Cleavage; D017931:DNA Primers / Q000737:chemistry*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D055668:Photochemical Processes; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D004247:DNA; D004259:DNA-Directed DNA Polymerase