SSB Enhances Detection of ssDNA Translocation through Solid-State Nanopores

The small diameter and secondary structure formation are major problems in nanopore-based analysis of hetero-sequence ssDNA/RNA. Here we report how binding of single-stranded binding protein (SSB) can both prevent secondary formation and increase diameter of ssDNA. SSB is a helix-destabilizing protein in virtue of its binding with high affinity to ssDNA and plays important roles in DNA replication, recombination and repair. E.coli SSB forms tetramers and binds every 35 nucleotides (nt) under conditions used in our experiments. We have translocated long (7.2 kb) and short SSB-coated ssDNA in the 37-100 nt range. For long SSB-coated ssDNA, current blockade levels are lower and last significantly longer than those of the free ssDNA, which is due to straightening of the globular structure. SSB-coated ssDNA molecules as short as 37-100 nt translocate much faster but are still easily detectable. We found translocation times of 0.92±0.18 ms for 37-nt ssDNA/SSB and 1.40±0.14 ms for 100-nt SSB/ssDNA. This is the first demonstration that ssDNA shorter than 100 nt can be detected using solid-state nanopore and applicable for future applications, such as ssDNA sizing or sequencing of the natural and long ssDNA, which forms complicated secondary structure.View Large Image | View Hi-Res Image | Download PowerPoint Slide