Design and synthesis of mirror image fluorescent nucleotide reversible terminator, (L)-3'-O-azidomethyl-dGTP-N3-fluorophore, and (D)-9°N mutant DNA polymerase for mirror image (L)-DNA sequencing by synthesis

Abstract

Functional DNA molecules such as aptamers, catalytic DNAzymes, and dynamic nanostructures have many promising applications in diagnostics and therapeutics yet suffer from limited utility due to immunogenicity and unwanted interactions with other biomolecules, leading to nuclease degradation and unintended sequence driven binding to native sample DNA. Bio-orthogonal, (L)-form or mirror image DNA overcome these challenges yet suffers from the inability to sequence these molecules in a high-throughput manner, a necessary step in the rapid creation of functional (L)-DNA. Here, we report for the first time, design, synthesis, and functional demonstration of (L)-DNA sequencing by synthesis (SBS) using a fluorescent nucleotide reversible terminator, (L)-3'-O-azidomethyl-dGTP-N3-fluorophore paired with a (D)-9{degrees}N mutant DNA polymerase, both enantiomers (i.e., mirror image) of their natural biologically active forms. TABLE OF CONTENTS (GRAPHICAL ABSTRACT) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/692211v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@160650forg.highwire.dtl.DTLVardef@18add51org.highwire.dtl.DTLVardef@1c80104org.highwire.dtl.DTLVardef@1bad99b_HPS_FORMAT_FIGEXP M_FIG C_FIG