Study on Suitability of KOD DNA Polymerase for Enzymatic Production of Artificial Nucleic Acids Using Base/Sugar Modified Nucleoside Triphosphates

Abstract

Recently, KOD and its related DNA polymerases have been used for preparing various modified nucleic acids, including not only base-modified nucleic acids, but also sugar-modified ones, such as bridged/locked nucleic acid (BNA/LNA) which would be promising candidates for nucleic acid drugs. However, thus far, reasons for the effectiveness of KOD DNA polymerase for such purposes have not been clearly elucidated. Therefore, using mutated KOD DNA polymerases, we studied here their catalytic properties upon enzymatic incorporation of nucleotide analogues with base/sugar modifications. Experimental data indicate that their characteristic kinetic properties enabled incorporation of various modified nucleotides. Among those KOD mutants, one achieved efficient successive incorporation of bridged nucleotides with a 2′-ONHCH2CH2-4′ linkage. In this study, the characteristic kinetic properties of KOD DNA polymerase for modified nucleoside triphosphates were shown, and the effectiveness of genetic engineering in improvement of the enzyme for modified nucleotide polymerization has been demonstrated.

In vitro selection of BNA (LNA) aptamers

“…[48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65] Veedu et al first tested B/L nucleotide incorporation using Phusion High Fidelity DNA polymerase, 9°Nm DNA polymerase, and Pfu DNA polymerase, and observed that the first and second of these three polymerases to exhibit superior stability in human plasma and target binding affinity (t 1/2 = 53 h, EC 50 = 2.0 nM) compared with TTA1 (t 1/2 = 42 h, EC 50 = 5.8 nM). In contrast, replacement with 2'-OMe nucleotides at the same positions, which yielded TTA1.1, resulted in a more than 2-fold decrease in binding affinity, although stability was substantially improved (t 1/2 = 49 h, EC 50 = 13.7 nM).…”

“…84,85 This approach has been successfully implemented and was published in Science in 2012, and a variant of Tgo DNA polymerase derived from Thermococcus gorgonarius, PolC7, was created. 26 Another way is to design polymerase variants and assess their catalytic abilities one by one; 56 we discovered several KOD variants suitable for BNA syntheses in our collaboration with Toyobo Co., Ltd. These polymerases are promising candidates for applications in BNA aptamer selection and will be used as parent enzymes for further improvements.…”

“…In 2',4'-BNA/LNA syntheses, KOD Dash DNA polymerase was used for library A-D selections, while a 19:1 enzyme mix of KOD Dash and KOD mutant DNA polymerases (KOD2) 56 was used for library E selection. Unlike in library A-D selections, obvious complex peaks could not be observed in library E selection, and electrograms seemed to remain unchanged during the course of selection (Fig.…”

Selection of LNA-containing DNA aptamers against recombinant human CD73

“…Therefore in investigating the properties of LNA nucleotides in aptamers, we and others, have focused on selecting aptamers using SELEX methods, front-loaded with LNA and other modified nucleotides. [19][20][21][22][23] Commercially available polymerases are capable of reading and incorporating LNA nucleotides, 22,24,25 but the efficacy of such reactions is often low. Despite this, Kasahara et al 19 recently reported the selection of aptamers containing LNA nucleotides in the primer and the random region.…”

DNA display of folded RNA libraries enabling RNA-SELEX without reverse transcription

“…To improve chemical diversity, base-modified DNA aptamers have been readily utilized, largely owing to the discovery that family B DNA polymerases (including Vent, KOD, and Pfu) can accommodate C5-substituted thymidine base analogs. 3, 11–15, 18–22 In particular, SOMAmer technology, which incorporates short hydrophobic groups at the C5 position of uridine, improved the success rate of obtaining high-affinity aptamers from 30% to 80% for hundreds of targets. 20 Recently, our group reported a method termed SELection of Modified Aptamers, or SELMA, allowing for the incorporation of large modifications into DNA libraries, which was successfully used to obtain multivalent glycoclusters that mimic a conserved epitope on the HIV envelope protein gp120.…”