Rapid incorporation kinetics and improved fidelity of a novel class of 3'-OH unblocked reversible terminators

Abstract

Recent developments of unique nucleotide probes have expanded our understanding of DNA polymerase function, providing many benefits to techniques involving next-generation sequencing (NGS) technologies. The cyclic reversible termination (CRT) method depends on efficient base-selective incorporation of reversible terminators by DNA polymerases. Most terminators are designed with 3'-O-blocking groups but are incorporated with low efficiency and fidelity. We have developed a novel class of 3'-OH unblocked nucleotides, called Lightning Terminators™, which have a terminating 2-nitrobenzyl moiety attached to hydroxymethylated nucleobases. A key structural feature of this photocleavable group displays a 'molecular tuning' effect with respect to single-base termination and improved nucleotide fidelity. Using Therminator DNA polymerase, we demonstrate that these 3'-OH unblocked terminators exhibit superior enzymatic performance compared to two other reversible terminators, 3'-O-amino-TTP and 3'-O-azidomethyl-TTP. Lightning Terminators show maximum incorporation rates (k(pol)) that range from 35 to 45 nt/s, comparable to the fastest NGS chemistries, yet with catalytic efficiencies (k(pol)/K(D)) comparable to natural nucleotides. Pre-steady-state kinetic studies of thymidine analogs revealed that the major determinant for improved nucleotide selectivity is a significant reduction in k(pol) by >1000-fold over TTP misincorporation. These studies highlight the importance of structure-function relationships of modified nucleotides in dictating polymerase performance.

Figure 1.

Chemical structures of (A) 3′-OH unblocked reversible terminators: dU.V and dU.VI, (B) 3′-O-modified reversible terminators: 3′-O-amino-TTP and 3′-O-azidomethyl-TTP and (C) 3′-OH unblocked reversible terminators: dA.VI, dC.VI and dG.VI. Red chemical structures denote terminating functional groups that cleave when exposed to (A and C) UV light or (B) sodium nitrite or tris(2-carboxyethyl)phosphine, respectively.

Figure 2.

Burst kinetics plots of (A) dA.VI, (B) dC.VI, (C) dG.VI and (D) dU.VI. See experimental section for details.

Figure 3.

Single-base termination of dU.V and dU.VI. Therminator DNA polymerase was bound to primer/template complex in 1× Thermopol buffer and subjected to 10 min incubation at 75°C in HPLC water (lane ‘P’ or primer), 50 nM TTP (lane ‘Ct’ or control) or 10 μM dU.V followed by a 90 s exposure to 365 nm UV light (lane ‘Pc’ or photochemical cleavage)—control lanes demonstrating that the incorporation bands of dU.V and dU.VI are the result of a single-base incorporation event. The remaining lanes represent incubation time courses of 10 μM dU.V or 10 μM dU.VI. Prior to performing the termination assays, dU.V and dU.VI were purified using the mop-up assay to remove trace amounts of HOMedUTP (43). Weighted-sum analysis was performed, yielding a value = 1.0 (white asterisks) at time points from 2 to 20 min (38). We note that the weighted-sum method, which has been reported previously (38), is used to measure quantitatively the termination property of nucleotide analogs being extended along a homopolymer stretch of complementary template bases. The slightly slower mobility of dU.VI termination products over those of dU.V results from the higher mass of the OMe group. Assays were performed in triplicate, a representative gel of which is shown.

Figure 4.

Models of (A) dU.VI and (B) 3′-O-azidomethyl-TTP interactions with Therminator DNA polymerase. The RB69 DNA polymerase ternary structure [PDB ID: 1IG9; see reference (65)] is used as a structural model for Therminator DNA polymerase and its active site [from 9°N-7 DNA polymerase (58)] was aligned with bound TTP using the pair fit function in MacPyMol (The PyMOL Molecular Graphics System, Version 1.5.0.1 Schrödinger, LLC). (A) dU.VI and (B) 3′-O-azidomethyl-TTP were drawn as extension of the three-dimensional structural model for TTP. Dotted lines in (A) represent hydrogen bonds between the 3′-OH group of dU.VI and its non-bridging oxygen of the β-phosphate group and –NH group of the peptide bond for Y409 (Therminator DNA polymerase amino acid numbering) that illustrate proper geometric alignment of these groups for efficient catalysis. The extension of the 3′-O-azidomethyl group in (B) is expected to cause a steric clash with the phenolic group side chain of Y409, thereby abolishing its incorporation efficiency. Improved incorporation performance with Therminator III, which contains the three substitutions (L408S, Y409A and P410V) that reduce the size of each amino acid side chain, supports this model.