Biomedical subjects
Publications and source records attributed to Rohan T Ranasinghe.
Synthetic chemistry has been central to the design of modern methods of genetic analysis. In this article, we discuss the underlying chemistry and biophysical principles that have been used in the development of robust methods for the analysis of DNA in the diagnostic laboratory.
We have achieved recognition of all 4 bp by triple helix formation at physiological pH, using triplex-forming oligonucleotides that contain four different synthetic nucleotides. BAU [2'-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine] recognizes AT base pairs with high affinity, (Me)P (3-methyl-2 aminopyridine) binds to GC at higher pHs than cytosine, while (A)PP (6-(3-aminopropyl)-7-methyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one) and S [N-(4-(3-acetamidophenyl)thiazol-2-yl-acetamide)] bind to CG and TA base pairs, respectively. Fluorescence melting and DNase I footprinting demonstrate successful triplex formation at a 19mer oligopurine sequence that contains two CG and two TA interruptions. The complexes are pH dependent, but are still stable at pH 7.0. BAU, (Me)P and (A)PP retain considerable selectivity, and single base pair changes opposite these residues cause a large reduction in affinity. In contrast, S is less selective and tolerates CG pairs as well as TA.
Substituted 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleoside analogues have been synthesised from 5-alkynyl-uridine derivatives, incorporated into triplex forming oligonucleotides (TFOs) and found to selectively bind CG inversions with enhanced affinity compared to T.