Site-Specific Enzymatic Syntheses of DNA Bearing Several Modifications From RNA or DNA Templates

Two approaches for the enzymatic synthesis of DNA oligonucleotides (ONs) bearing multiple site-specific modifications were explored. The first approach based on repeated single nucleotide extension (SNI) and reverse transcription from RNA template with Bst 3.0 DNA polymerase followed by digestion and exchange of template efficiently produced an ON with single modification, but the second round gave mixtures of full-length and truncated products. The second approach was based on repeated cycles of SNI and primer extension from a DNA template followed by λ exonuclease digestion and exchange of the template and it worked very well even for consecutive incorporation of three different base-modified nucleotides at specific positions. This was demonstrated on the synthesis of a molecular beacon bearing Cy3 and Cy5 fluorophores for the FRET detection of target RNA. The method is an efficient single-tube operation that does not require separation and purification of intermediates and gives clean ONs labeled at desired positions.