Mass-spectrometry analysis of modifications at DNA termini induced by DNA polymerases

Abstract

Non-natural nucleotide substrates are widely used in the enzymatic synthesis of modified DNA. The terminal activity of polymerases in the presence of modified nucleotides is an important, but poorly characterized, aspect of enzymatic DNA synthesis. Here, we studied different types of polymerase activity at sequence ends using extendable and non-extendable synthetic models in the presence of the Cy5-dUTP analog Y. In primer extension reactions with selected exonuclease-deficient polymerases, nucleotide Y appeared to be a preferential substrate for non-templated 3′-tailing, as determined by MALDI mass-spectrometry and gel-electrophoresis. This result was further confirmed by the 3′-tailing of a non-extendable hairpin oligonucleotide model. Additionally, DNA polymerases induce an exchange of the 3′ terminal thymidine for a non-natural nucleotide via pyrophosphorolysis in the presence of inorganic pyrophosphate. In primer extension reactions, the proofreading polymerases Vent, Pfu, and Phusion did not support the synthesis of Y-modified primer strand. Nevertheless, Pfu and Phusion polymerases were shown to initiate terminal nucleotide exchange at the template. Unlike non-proofreading polymerases, these two enzymes recruit 3′–5′ exonuclease functions to cleave the 3′ terminal thymidine in the absence of pyrophosphate.

Evaluation of 3′-phosphate as a transient protecting group for controlled enzymatic synthesis of DNA and XNA oligonucleotides

“…Intrigued by these results, we analyzed the products stemming from the PEX reactions conducted with the combination of Taq polymerase and 3′-phos-dTTP 5 as well as that with Therminator and 3′-phos-LNA-TTP 10 by LCMS (Table 1 , Supplementary Figs. 23 – 28 , and Supplementary Note 1 ) using established protocols for products stemming from PEX reactions with natural and modified nucleotides 51 , 52 . This analysis clearly revealed that 1. no phosphorylated nucleotide was incorporated by polymerases and that 2. dA nucleotides were misincorporated opposite templating dAs instead of the modified triphosphates.…”

Anticoagulant Oligonucleotide–Peptide Conjugates: Identification of Thrombin Aptamer Conjugates with Improved Characteristics

“…The preparation of thymidine phosphoramidite derivative 2 with activated carboxylic acid residue at the N3 position of the pyrimidine heterocycle is outlined in Scheme 1 . The synthesis of 5′-O-(4,4′-dimethoxytrityl)-N3-carboxymethylthymidine was carried out as described previously by selective N-alkylation of the partially protected thymidine with ethyl bromoacetate in the presence of DBU, followed by alkaline hydrolysis [ 6 , 17 ]. Further activation of the carboxyl group smoothly proceeded upon treatment with an excess of pNP-TFA ester in pyridine [ 18 ].…”