DNA Polymerase I Large Fragment from Deinococcus radiodurans, a Candidate for a Cutting-Edge Room-Temperature LAMP

Abstract

In recent years, the loop-mediated isothermal amplification (LAMP) technique, designed for microbial pathogen detection, has acquired fundamental importance in the biomedical field, providing rapid and precise responses. However, it still has some drawbacks, mainly due to the need for a thermostatic block, necessary to reach 63 °C, which is the BstI DNA polymerase working temperature. Here, we report the identification and characterization of the DNA polymerase I Large Fragment from Deinococcus radiodurans (DraLF-PolI) that functions at room temperature and is resistant to various environmental stress conditions. We demonstrated that DraLF-PolI displays efficient catalytic activity over a wide range of temperatures and pH, maintains its activity even after storage under various stress conditions, including desiccation, and retains its strand-displacement activity required for isothermal amplification technology. All of these characteristics make DraLF-PolI an excellent candidate for a cutting-edge room-temperature LAMP that promises to be very useful for the rapid and simple detection of pathogens at the point of care.

Keywords: DNA polymerase; DNA replication; Deinococcus radiodurans; LAMP; extremophiles.

Figure 1

AlphaFold2 structure of D. radiodurans DNA Polymerase I. The Alphafold model of DNA polymerase I P52027 (DPO1_DEIRA) is reported in the figure. The poorly structured N-terminal region, aa 1–37, has been removed. In grey, the large region (38–361 aa) deleted in the construct used in our study including the nuclease domain and a partial 3′–5′ exonuclease domain is shown. In magenta, there is the evolutionarily conserved Motif A. The other colors are dictated by different confidence levels: dark blue: very high (pLDDT > 90); light blue: high (90 > pLDDT > 70); yellow: low (70 > pLDDT > 50); orange: very low (pLDDT < 50).

Figure 2

Analysis of DraLF-PolI DNA polymerase activity at different temperatures. (A) Increasing amounts of DraLF-PolI (from 0.05 up to 5 pmoles) were analyzed for their DNA polymerase activity on PE substrate (obtained as described in Section 4). The reactions were performed at the indicated temperatures. Lane 1 represents the control reaction where DraLF-PolI was omitted. (B) Graphic representation of the DNA polymerase assays at the indicated temperatures. The values obtained are the average of three different experiments.

Figure 3

Thermostability analysis of DraLF-PolI. In total, 3 pmoles of of DraLF-PolI were preincubated at the indicated different temperatures for increasing times (from 0 up to 60 min) (A–D). The residual activity of the enzyme was tested at 30 °C on PE substrate after 10, 20, 30 and 60 min (lanes 3–6 and 7–10). Lane 1 refers to the negative control in which the substrate was incubated for 60 min in the absence of protein. Lane 2 is the positive control where the protein was not preincubated. (E) Graphic representation of DraLF-PolI thermostability at the indicated temperatures. The values are the average of three different experiments.

Figure 4

DNA Polymerase activity time-course at 60 °C. The activity of DraLF-PolI (3 pmoles) was tested at 60 °C, on a PE substrate, at increasing times, from 5 to 240 s (lanes 2–7). The negative control was performed in the absence of enzyme (lane 1).

Figure 5

CD analyses. (A): Far-UV CD spectrum recorded at 20 °C. (B): Thermal denaturation monitored at 220 nm. In the insert, spectra registered at different temperatures.

Figure 6

DraLF-PolI selectivity for dNTPs over time. (A–D) DNA polymerase assays without dGTP, dATP, dTTP and dCTP, respectively. In total, 3 pmoles of DraLF-PolI were incubated at 30 °C in the absence of one of the 4 dNTPs, at increasing times, as indicated (lanes 2–8). Lane 9 corresponds to the positive control in which the reactions were carried out in the presence of all dNTPs. Negative controls were performed in the absence of protein (lane 1).

Figure 7

Incorporation of a single dNTP over time. (A–D) DNA polymerase assays with dGTP, dATP, dTTP or dCTP, respectively. In total, 3 pmoles of DraLF-PolI were incubated in the presence of only one of the four nucleotides, at increasing times, as indicated (lanes 2–8). Lane 9 correspond to the positive control in which the reactions were carried out in the presence of all dNTPs. Negative controls were performed in the absence of protein (lane 1).

Figure 8

Strand-displacement activity assays. DraLF-PolI polymerase activity was tested on PE and SD substrates. (A) Schematic representation of the substrate used in this experiment, lanes 6–10. (B) DNA polymerase vs Strand-Displacement assays. A negative control was carried out in the absence of enzyme (lanes 1 and 6). Increasing amounts of DraLF-PolI (lanes 2–5; lanes 7–10) were analyzed.

Figure 9

Strand-displacement activity assays of DraLFPolI on SDgap0 and Sdgap1 substrate. DraLF-PolI polymerase activity was tested on a substrate with 1 nucleotide gap (A) and on a substrate with no gap (B). Strand-displacement activity is observed in the presence of increasing quantities of DraLF-PolI (0.01 at 5 pmoles, as indicated); the positive controls (lanes 9, (A,B)) were performed on the PE substrate in order to unequivocally identify the full-length product (lanes 2–8, panels (A,B)). A negative control was performed in the absence of enzyme (lanes 1, panels (A,B)).

Figure 10

DraLF-PolI strand-displacement activity assays at increasing temperatures. The strand-displacement activity is observed in the presence of increasing amounts of protein (0.01 to 5 pmoles) (lanes 2–8, panels (A–D)). Negative controls were performed in the absence of proteins (lanes 1, panels (A–D)); positive controls (lanes 9, panels (A–D)) were performed with PE substrate. The temperatures analyzed are indicated.

Figure 11

DNA binding affinity of DraLFPolI with different DNA structures. Electrophoretic mobility shift assays were performed using different DNA substrates as described in Methods to estimate binding affinity of DraLFPolI. Binding affinity to ssDNA 21mer (A) and 45mer (B); (C) binding affinity to a primed DNA structure; (C) binding affinity to a gapped dsDNA; and (D) binding affinity to a dsDNA. The assays were performed in the presence of increasing amounts of protein from 0.5 to 40 pmoles, as indicated (lanes 2–8, panels (A–E)). The negative controls were performed in the absence of protein (lane 1, panels (A–E)).

Figure 12

Analysis of DraLF-PolI activity at different pHs. (A) DNA polymerase activity of DraLF-PolI was evaluated at different pH. In total, 3 pmoles of enzyme were incubated in the presence of PE substrate as described in Section 4. (B) Selected buffers. The negative controls (-) were performed in the absence of protein.

Figure 13

Analysis of DraLF-PolI activity under different storage conditions. DraLF-PolI polymerase activity was tested on PE substrate as described in Section 4, under different storage conditions. (A) Residual activity 24 h after desiccation, storage at room temperature and rehydration; (B) residual activity 24 h after storage in a liquid state at room temperature; (C) residual activity 24 h after storage in a liquid state at 4 °C. The assays were performed in the presence of increasing amounts of protein from 0.04 to 12 pmoles, as indicated (lanes 2–6, panels (A–C)). The negative controls were performed in the absence of protein (lane 1, panels (A–C)).

Figure 14

Substrate structure used in this work.