Enzymatic Incorporation of LNA Nucleotides into DNA Strands

Abstract

Unlocking uses of locked nucleic acids: LNA nucleoside 5′‐triphosphates have been synthesized, and their ability to serve as substrates for polymerases have been investigated. Phusion high‐fidelity DNA polymerase was found to be an efficient enzyme for incorporating LNA nucleoside 5′‐triphosphates into DNA strands.

Smart functional nucleic acid chimeras: Enabling tissue specific RNA targeting therapy

“…[135][136][137] Toward developing LNA-modified aptamers, Veedu et al reported the enzymatic recognition capabilities of LNA nucleotides using DNA and RNA polymerases. [138][139][140][141][142][143][144] In 2013, Kuwahara and co-workers www.tandfonline.comreported an LNA (BNA) aptamer against thrombin using capillary electrophoresis-based SELEX (CE-SELEX) method. 145,146 …”

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).…”

Polymerase-directed synthesis of C5-ethynyl locked nucleic acids

“…For this purpose, we synthesized LNA-U and C5-ethynyl LNA-U nucleoside 5 0 -triphosphates (Fig. 2) from known intermediates 12,15 using protocols similar to those previously described 16 (see Supplementary data for details). To investigate the substrate specificity of LNA-UTP and C5-ethynyl LNA-UTP for polymerases, we designed primer and template DNA sequences to perform primer extension (DNA synthesis using DNA polymerase) and transcription reactions ( First, we tested LNA-U nucleoside triphosphate as a substrate for T7 RNA polymerase in transcription reactions in vitro.…”

“…[16][17][18][19][20][21] This motivated us to examine LNA-U nucleoside 5 0 -triphosphates as substrates for DNA polymerases. In this experiment, a 19nt long 5 0 -32 P labeled primer DNA (P1, Fig.…”

“…These findings are in line with our previous report on enzymatic incorporation of consecutive LNA-T nucleotides. 16 We have also investigated the incorporation of C5-ethynyl LNA-U nucleotides into RNA strands using T7 RNA polymerase. The experiment revealed that unlike LNA-U nucleotides, T7 RNA polymerase failed to accept C5-ethynyl LNA-U nucleoside triphosphate as a substrate (data not shown).…”