Measuring cation dependent DNA polymerase fidelity landscapes by deep sequencing

Area of Science:

• Biotechnology
• Molecular Biology
• Nanotechnology

Background:

• High-throughput signal recording in inaccessible micro-environments is challenging.
• A nanoscale recording device is needed for quantitative transduction of variables into molecular data.
• DNA polymerases (DNAPs) can potentially encode local cation concentration via misincorporation rates.

Purpose of the Study:

• To quantify the cation sensitivity of DNAP misincorporation rates.
• To enable indirect readout of cation concentration through DNA sequencing.
• To characterize DNAP misincorporation for developing molecular recording devices.

Main Methods:

• Multiplexed deep sequencing was used to quantify misincorporation properties of Dpo4 and Klenow exo(-) DNA polymerases.
• The probability and base selectivity of misincorporation across template positions were determined.
• Cation (Mn2+, Mg2+) dependence of misincorporation was analyzed.

Main Results:

• Dpo4 functions as a DNA recording device for Mn2+, exhibiting a misincorporation rate gain of ~2%/mM.
• Misincorporation modulation is template base-selective; Dpo4 shows >50-fold increase on template T.
• Cations act as scaling factors, altering overall misincorporation rates without changing relative frequencies of incorporated nucleotides.

Conclusions:

• Characterization of ion dependence is the first step toward repurposing DNAP as a molecular recording device.
• This approach enables indirect readout of cation concentrations via DNA sequencing.
• The findings pave the way for novel nanoscale sensors for micro-environmental monitoring.