Nanopores are being used for analyzing the behavior of biomolecules, such as proteins, RNA and DNA. However, interference between proteins utilized as nanopores and the test molecules is a major issue for the quantitative analysis.
A research team led by Professor Ulrich Gerland from Ludwig-Maximilians-Universitat Munchen (LMU) and Professor Friedrich Simmel from Technische Universitat Munchen has devised a novel approach based on the study of reverse translocation via non-symmetric pores to reduce the interactions of nanopores with test molecules, paving the way to study complex polynucleotides’ secondary structures.
With this method, the team was able to quantitatively illustrate the path of simple-structured polynucleotide sequences via nanopores. The method has also helped the team to create a theoretical model to determine the dynamics of translocation of nucleic acids differing in their nucleotide sequences, using thermodynamic calculations of ‘free-energy landscapes.’
Polynucleotides such as RNA and DNA are capable of folding to form their secondary structures, which manipulate their biochemical functions. According to the novel method, measurements are made on molecules when they translocate via the nanopore backward. As the first step, the polynucleotide to be analyzed is made to enter via the conical opening from one side due to an electrical potential, causing the unfolding of the secondary structure of the polynucleotide. The molecule refolds when it comes out. The polynucleotide chain cannot completely pass through to the other side of the nanopore due to the presence of an anchor at its one end. The potential is reversed during the return journey of the polynucleotide through the nanopore, starting the unfolding process at the pore’s narrow end as well the analysis.
Gerland explained that the polynucleotide sequence gets unfolded when it travels via narrow nanopores, and translocation dynamics reveal the molecule’s structural features without using a fluorescent label.