5′-DMT-protected double-stranded DNA: Synthesis and competence to enzymatic reactions

Abstract

The functionalization of 5ʹ-OH group in nucleic acids is of significant value for molecular biology. In the current work we discovered that acid-labile 4,4ʹ-dimethoxytrityl protecting group (DMT) of oligonucleotides (ONs) is stable under PCR conditions and does not interfere with activity of DNA polymerases. So application of 5ʹ-DMT-protected ONs could allow producing both symmetric and asymmetric 5ʹ-DMT-blocked double-stranded DNA (dsDNA) fragments. We demonstrated that the presence of thiol compounds (mercaptoethanol and dithiothreitol) in PCR mixture is undesirable for the stability of DMT-group. DMT-ONs can be successfully used during polymerase chain assembly of synthetic genes. We tested 5ʹ-DMT dsDNA in blunt-end DNA ligation reaction by T4 DNA ligase and found that it could not be ligated with 5ʹ-phosphorylated DNA fragments, namely linearized plasmid vector pJET1.2/blunt. Possible reason for this is steric hindrance created by bulky and rigid DMT-group, that prevents entering enzyme active site. We also demonstrated that 5ʹ-DMT modification of dsDNA does not affect activity of T5 5ʹ,3ʹ-exonuclease towards both ssDNA and dsDNA. Further screening of the exonucleases, sensitive to 5ʹ-DMT-modification or search of ways to separate long 5ʹ-DMT-ssDNA and 5ʹ-OH-ssDNA could allow finding application of 5ʹ-DMT-modified oligo- and polynucleotides.