Docking and Activity of DNA Polymerase on Solid-State Nanopores

Area of Science:

• Nanotechnology
• Biophysics
• Molecular Biology

Background:

• Integration of motor enzymes with biological nanopores is key to DNA sequencing.
• Studies applying this principle to solid-state nanopores are limited.
• Solid-state nanopores offer potential advantages for sensing applications.

Purpose of the Study:

• To demonstrate real-time monitoring of phi29 DNA polymerase (DNAP) docking onto solid-state nanopore arrays.
• To investigate electrical and optical readout methods for detecting enzyme-nanopore interactions.
• To establish a proof-of-concept for utilizing enzyme docking in nanopore sensing.

Main Methods:

• Fabrication of sub-10 nm truncated-pyramidal nanopore (TPP) arrays using atomic layer deposition of hafnium oxide.
• Ionic current measurements on single TPPs to observe DNAP-DNA complex docking and translocation.
• Label-free optical detection using Ca2+ sensitive dye on TPP arrays to monitor DNAP docking dynamics.
• On-site rolling circle amplification to assess the activity of docked DNAP.

Main Results:

• Successful reduction of TPP pore size to sub-10 nm for enzyme interaction.
• Observation of temporal docking of polymerase-DNA complexes onto single TPPs.
• Demonstration of a label-free optical method for detecting DNAP docking on TPP arrays.
• Proof-of-concept for on-site DNA synthesis by docked DNAP.

Conclusions:

• phi29 DNA polymerase can be monitored docking onto solid-state nanopores using electrical and optical techniques.
• Label-free optical detection is effective for observing enzyme docking dynamics on nanopore arrays.
• The developed docking scheme shows promise for advanced nanopore sensing applications, including on-site DNA synthesis.