1. Self-Priming Enzymatic Fabrication of Multiply Modified DNA
- Author
-
Bernard A. Connolly, Rachel C. Little, Eimer Tuite, Kasid Khan, Andrew R. Pike, Colette J. Whitfield, and Kuniharu Ijiro
- Subjects
Stereochemistry ,DNA polymerase ,010402 general chemistry ,01 natural sciences ,Polymerase Chain Reaction ,Catalysis ,Nucleobase ,law.invention ,chemistry.chemical_compound ,law ,Nucleotide ,DNA slippage ,Polymerase ,Polymerase chain reaction ,chemistry.chemical_classification ,biology ,Base Sequence ,010405 organic chemistry ,Chemistry ,Nucleotides ,Organic Chemistry ,General Chemistry ,DNA ,0104 chemical sciences ,Enzyme ,Duplex (building) ,Modified DNA ,biology.protein ,Nucleic Acid Conformation ,Small molecule binding - Abstract
The self-priming synthesis of multiply modified DNA by the extension of repeating unit duplex “oligoseeds” provides a source of versatile DNA. Sterically-demanding nucleotides 5-Br-dUTP, 7-deaza-7-I-dATP, 6-S-dGTP, 5-I-dCTP as well as 5-(octadiynyl)-dCTP were incorporated into two extending oligoseeds; [GATC]5/[GATC]5 and [A4G]4/[CT4]4. The products contained modifications on one or both strands of DNA, demonstrating their recognition by the polymerase as both template (reading) and substrate (writing). Nucleobase modifications that lie in the major groove were reliably read and written by the polymerase during the extension reaction, even when bulky or in contiguous sequences. Repeat sequence DNA over 500 bp long, bearing four different modified units was produced by this method. The number, position and type of modification, as well as the overall length of the DNA can be controlled to yield designer DNA that offers sequence-determined sites for further chemical adaptations, targeted small molecule binding studies, or sensing and sequencing applications.
- Published
- 2018