Cognate base-pair selectivity of hydrophobic unnatural bases in DNA ligation by T4 DNA ligase

Abstract

We present cognate base pair selectivity in template-dependent ligation by T4 DNA ligase using a hydrophobic unnatural base pair (UBP), Ds-Pa. T4 DNA ligase efficiently recognizes the Ds-Pa pairing at the conjugation position, and Ds excludes the noncognate pairings with the natural bases. Our results indicate that the hydrophobic base pairing is allowed in enzymatic ligation with higher cognate base-pair selectivity, relative to the hydrogen-bond interactions between pairing bases. The efficient ligation using Ds-Pa can be employed in recombinant DNA technology using genetic alphabet expansion, toward the creation of semi-synthetic organisms containing UBPs.

Keywords: T4 DNA ligase; genetic alphabet expansion; ligation; unnatural base pair.

FIGURE 1

Enzymatic ligation by T4 DNA ligase using DNA with hydrophobic UBPs. A, T4 DNA ligase domain organization and the schematic representation of enzymatic ligation. The N‐terminal domain (NTD, pink) corresponds to the DNA‐binding domain (DBD). The nucleotidyl transferase domain (NTase, yellow) contains the catalytic core, and OBD (orange) is the oligonucleotide‐binding domain. The DNA duplex sequences and the complex structure of T4 DNA ligase with the DNA duplex, containing the adenylated DNA intermediate (AppDNA), were adopted from PDB: 6DT1. B, Enzymatic ligation reactions, where the UBP (X‐Y; e.g., Ds‐Pa) is located at the ligation junction (nick site). Chemical structures of A‐T and A‐F (C), Ds‐Pa and Ds‐Px (D), and A‐Pa (E). The minor groove hydrogen bond acceptor residues are indicated in solid circles (pink). The 2‐fluorine residue, which has lower hydrogen‐bond acceptor ability, is indicated in an open circle (pink)

FIGURE 2

Ligation of (A) 5′‐phosphorylated R18X (donor strand, X = Ds or A) to FAM‐L22 (acceptor strand) and (B) 5′‐ phosphorylated R17 (donor strand) to FAM‐L23X (acceptor strand, X = Ds or A) in the presence of Template 25 (Y = Pa, T, C, A, or G). Reaction conditions: 0.05 Weiss U/μL T4 DNA ligase (corresponding to 1/5 of the amounts of ligase used in Figures 2 and 3), 0.5 μM each DNA, 10 minutes at 22 °C. The FAM‐labeled DNA bands in the gel were detected with an LAS4000 bio‐imager. The ligated products are 40‐mers. The DNA species higher than 40‐mer are expected to be nontemplate ligated products (with one extra donor strand, since we did not block the 3′‐end)

FIGURE 3

Time course of the ligation reactions. A, Ligation of 5′‐phosphorylated R18X (donor strand) to FAM‐L22 (acceptor strand) in the presence of Template 25. B, Ligation of 5′‐phosphorylated R17 (donor strand) to FAM‐L23X (acceptor strand) in the presence of Template 25. X = Ds or A; Y = Pa, T, C, A, or G. Reaction conditions: 0.0025 Weiss U/μL T4 DNA ligase, 0.5 μM of each DNA at 22 °C. The time course of the reactions with the various base pairs were assessed by performing the ligation reactions at various time intervals: 1.25, 2.5, 5, 10 and 20 minutes

FIGURE 4

Summary of ligation efficiencies with the hydrophobic unnatural Ds base at the 5′‐end of the donor strand or at the 3′‐end of the acceptor strands. The rates of the ligation (turnover numbers) were calculated from the yields of the ligated products at 5 minutes (dsDNA: 0.5 μM; T4 DNA ligase: 0.0025 Weiss U/μL, 3.6 ± 0.9 nM) by averaging independent repetitive data (n = 2‐4, Figures 3, S8 and S10), and indicated with the standard deviations

FIGURE 5

Ligation of 5′‐phosphorylated R23 (A) or R22 (B) (donor strand) to L23X (A) or L24XT (B) (acceptor strand) in the presence of Template 35 (template strand). A 17‐mer DNA (17‐guide) was added to ensure the sufficient length of the duplex formation for T4 DNA ligase recognition. Reaction conditions: 0.25 Weiss U/μL T4 DNA ligase, 0.5 μM of each DNA fragment, 10 minutes at 22 °C. X = Ds or A, Y = Pa, T, C, A, or G. The DNA species higher than 46‐mer and those between 35‐mer and 46‐mer are expected to be nontemplate ligated products (with one extra donor strand, since we did not block the 3′ end)