Fluorous Base‐Pairing Effects in a DNA Polymerase Active Site

Abstract

We describe selective "fluorous" effects in the active site of a DNA polymerase, by using nucleotide analogues whose pairing edges are perfluorinated. The 5'-triphosphate deoxynucleotide derivatives of DNA base analogues 2,3,4,5-tetrafluorobenzene ((F)B) and 4,5,6,7-tetrafluoroindole ((F)I), as well as hydrocarbon controls benzene (B) and indole (I), were synthesized and studied as substrates for the DNA Polymerase I Klenow fragment (KF exo-). Modified nucleotides were present in the DNA template or were supplied as nucleoside triphosphates in studies of the steady-state kinetics of single nucleotide insertion. When supplied opposite the non-natural bases in the template strand, the hydrophobic nucleoside triphosphates were incorporated by up to two orders of magnitude more efficiently than the natural deoxynucleoside triphosphates. The purine-like fluorinated indole nucleotide ((F)I) was the most efficiently inserted of the four hydrophobic analogues, with the most effective incorporation occurring opposite the pyrimidine-like tetrafluorobenzene ((F)B). In all cases, the polyfluorinated base pairs were more efficiently processed than the analogous hydrocarbon pairs. A preliminary test of polymerase extension beyond these pairs showed that only the (F)B base is appreciably extended; the inefficient extension is consistent with recently published data regarding other nonpolar base pairs. These results suggest the importance of hydrophobicity, stacking, and steric interactions in the polymerase-mediated replication of DNA base pairs that lack hydrogen bonds. These findings further suggest that the enhanced hydrophobicity of polyfluoroaromatic bases could be employed in the design of new, selective base pairs that are orthogonal to the natural Watson-Crick pairs used in replication.

Highly fluorinated amphiphilic molecules and self-assemblies with biomedical potential

“…Selective pairing of the fluorinated bases with one another in duplex DNA was observed. DNA replication has subsequently been examined using polyfluorinated nucleotide analogues as substrates [128]. A DNA polymerase active site was able to process the polyfluorinated nucleotides more efficiently than the corresponding hydrocarbon pairs, demonstrating hydrophobic selectivity of fluorinated bases for other fluorinated bases.…”

Perfluorocarbons: Life sciences and biomedical uses Dedicated to the memory of Professor Guy Ourisson, a true RENAISSANCE man.

“…A DNA polymerase active site was able to process the polyfluorinated base pairs more effectively than the analogous hydrocarbon pairs, demonstrating hydrophobic selectivity of polyfluorinated bases for other polyfluorinated bases. 23 Incorporation of non-natural polyfluorinated bases, capable of enhanced (or ''hyper''-) hydrophobic interactions, expands the genetic alphabet beyond the natural one used in replication, thus illustrating the emerging role of F-compounds in developing chemistry, including constitutional dynamic ''bio''chemistry that lies well ''beyond'' nature.…”

Fluorescent Nucleic Acid Analogues in Research and Clinical Diagnostics

“…The substitution resulted in bright probes which were still able to form complementary complexes. It was also demonstrated that polymerase enzymes can interact and use these nucleoside analogues (Kool 2000;Lai and Kool 2005;Krueger and Kool 2009). This discovery inspired further designing new bases and base pairs as alternative genetic code that vary in shape/size and testing whether they function as normal base pairs.…”