Unnatural imidazopyridopyrimidine:naphthyridine base pairs: selective incorporation and extension reaction by Deep Vent (exo- ) DNA polymerase

Abstract

In our previous communication we reported the enzymatic recognition of unnatural imidazopyridopyrimidine:naphthyridine (Im:Na) base pairs, i.e. ImO(N):NaN(O) and ImN(O):NaO(N), using the Klenow fragment exo(-) [KF (exo(-))]. We describe herein the successful results of (i) improved enzymatic recognition for ImN(O):NaO(N) base pairs and (ii) further primer extension reactions after the Im:Na base pairs by Deep Vent DNA polymerase exo(-) [Deep Vent (exo(-))]. Since KF (exo(-)) did not catalyze primer extension reactions after the Im:Na base pair, we carried out a screening of DNA polymerases to promote the primer extension reaction as well as to improve the selectivity of base pair recognition. As a result, a family B DNA polymerase, especially Deep Vent (exo(-)), seemed most promising for this purpose. In the ImO(N):NaN(O) base pair, incorporation of NaN(O)TP against ImO(N) in the template was preferable to that of the natural dNTPs, while incorporation of dATP as well as dGTP competed with that of ImO(N)TP when NaN(O) was placed in the template. Thus, the selectivity of base pair recognition by Deep Vent (exo(-)) was less than that by KF (exo(-)) in the case of the ImO(N):NaN(O) base pair. On the other hand, incorporation of NaO(N)TP against ImN(O) in the template and that of ImN(O)TP against NaO(N) were both quite selective. Thus, the selectivity of base pair recognition was improved by Deep Vent (exo(-)) in the ImN(O):NaO(N) base pair. Moreover, this enzyme catalyzed further primer extension reactions after the ImN(O):NaO(N) base pair to afford a faithful replicate, which was confirmed by MALDI-TOF mass spectrometry as well as the kinetics data for extension fidelity next to the ImN(O):NaO(N) base pair. The results presented in this paper revealed that the ImN(O):NaO(N) base pair might be a third base pair beyond the Watson-Crick base pairs.

Figure 1.

Structures of Im:Na base pairs consisting of four H-bonds.

Figure 2.

Primer extension reactions by KF (exo⁻). (A) The reaction for ImO^N:NaN^O base pair. (B) The reaction for ImN^O:NaO^N base pair. Lanes 2–9 used the primer-template combination 5′-FITC-GTTCTGGATGGTCAGCGCAC-3′ (20mer) and 3′-CAAGACCTACCAGTCGCGTGXGAACGGGTG-5′ (30mer, X = ImO^N, NaN^O, ImN^O or NaO^N). Lane 1 indicates the 20-mer primer, and lane 10 indicates the control 30mer prepared by primer extension reaction (X = T).

Figure 3.

Screening of DNA polymerases for single-nucleotide insertion. (A) The reaction for ImO^N:NaN^O base pair. (B) The reaction for ImN^O:NaO^N base pair. All reactions used the primer-template combination 5′-FITC-GTTCTGGATGGTCAGCGCAC-3′ (20mer) and 3′-CAAGACCTACCAGTCGCGTGXGAACGGGTG-5′ (30mer, X = ImO^N, NaN^O, ImN^O or NaO^N). Lanes 1 and 14 indicate the 20-mer primer; lanes 2, 8, 15 and 21 indicate the results of KF (exo⁻); lanes 3, 9 16, and 22 indicate those of Taq DNA polymerase; lanes 4, 10, 17 and 23 indicate those of Tth DNA polymerase; lanes 5, 11, 18 and 24 indicate those of Vent (exo⁻) DNA polymerase; lanes 6, 12, 19 and 25 indicate those of Deep Vent (exo⁻) DNA polymerase; and lanes 7, 13, 20 and 26 indicate those of KOD Dash DNA polymerase.

Figure 4.

Single-nucleotide insertion by Deep Vent (exo⁻) (selectivity toward natural dNTPs). (A) The reaction for ImO^N:NaN^O base pair. (B) The reaction for ImN^O:NaO^N base pair. All reactions used the primer-template combination 5′-FITC-GTTCTGGATGGTCAGCGCAC-3′ (20mer) and 3′-CAAGACCTACCAGTCGCGTGXGAACGGGTG-5′ (30mer, X = ImO^N, NaN^O, ImN^O or NaO^N). Lanes 1 and 14 indicate the 20-mer primer.

Figure 5.

Primer extension reactions by family B DNA polymerases. (A) The reaction for ImO^N:NaN^O base pair. (B) The reaction for ImN^O:NaO^N base pair. All reactions used the primer-template combination 5′-FITC-GTTCTGGATGGTCAGCGCAC-3′ (20mer) and 3′-CAAGACCTACCAGTCGCGTGXGAACGGGTG-5′ (30mer, X = ImO^N, NaN^O, ImN^O or NaO^N). The reaction was carried out in the presence of dYTP (NaN^OTP, ImO^NTP, NaO^NTP or ImN^OTP) and natural dNTPs. Lane 1 indicates the 20-mer primer; lane 2 indicates the control 30mer prepared by primer extension reaction (X = T); lanes 3, 5, 7 and 9 indicate the results of Vent (exo⁻); and lanes 4, 6, 8 and 10 indicate those of Deep Vent (exo⁻) DNA polymerase.

Figure 6.

MALDI-TOF mass spectrum of the full-length 30mer. The sequence of the sample is 5′-FITC-GTTCTGGATGGTCAGCGCACImN^OCTTGCCCAC-3′. The exact structure of the 30mer and original spectrum were given in the Supplementary Data (Figure S2).

Figure 7.

Structures of possible base pairs between NaN^O and natural bases.