Backing out of the nanotunnel: New method for nucleic acid analysis

Jan 10, 2012

Nanopores provide a versatile tool for probing molecular structures. A new German study shows that one can obtain more detailed information about the dynamic behavior of nucleic acids during passage through nanopores by directing them to asymmetric pores for the return journey.

In the world of biomolecules such as proteins and the hereditary nucleic acids DNA and RNA, three-dimensional structure determines function. Analysis of the passage of such molecules through nanopores offers a relatively new, but highly promising, technique for obtaining information about their spatial conformations. However, interactions between the test molecules and the proteins used as pores have so far hindered quantitative analysis of the behavior of even simply structured molecules within nanopores. This problem must be solved before the technique can be routinely used for . In a project carried out under the auspices of the Cluster of Excellence "Nanosystems Initiative Munich" (NIM), researchers led by LMU physicist Professor Ulrich Gerland and Professor Friedrich Simmel (Technical University of Munich) have developed a new method that depends on the analysis of reverse translocation through asymmetric pores, which minimizes the interference caused by interactions with the pore material itself. This approach has enabled the team to construct a theoretical model that allows them to predict the translocation dynamics of nucleic acids that differ in their nucleotide sequences.

The nucleic acids RNA and DNA both belong to the class of molecules known chemically as polynucleotides. Both are made up of strings of four basic types of building blocks called nucleotides, which fall into two complementary pairs. In their single-stranded forms, DNA and RNA can fold into what are called secondary structures, as complementary nucleotides in the sequence pair up, forcing the intervening segments to form loops. If the single-stranded loop is very short, the secondary structure is referred to as a hairpin. As in the case of proteins, the secondary structures of influence their biochemical functions. The elucidation of the secondary structure of nucleic acid sequences is therefore of great interest.

"Nanopores are increasingly being employed to investigate the secondary structures of RNA and DNA," Gerland points out. "Passage through narrow nanopores causes the sequence to unfold, and the dynamics of translocation provide insights into the structural features of the molecules, without the need to modify them by adding a fluorescent label. The technique is relatively new, and its potential has not yet been fully explored."

In the new study, he and his collaborators used a new experimental procedure, which allowed them to quantitatively describe the passage of simply structured polynucleotide sequences through nanopores, and develop a theoretical model that accounts for their findings. This level of understanding has not been achieved previously, because complicating factors such as interactions between the protein nanopore and the polynucleotide have had a significant influence on the measurements and made it difficult to predict the behavior of the test molecules.

Thanks to a clever change in experimental design, the impact of these factors has now been minimized. The trick is to perform the measurements on molecules as they translocate through the pore in reverse. First, the polynucleotide of interest is forced through the conical orifice from one side under the influence of an electrical potential. This causes its secondary structure to unfold and, as it emerges, the molecule refolds. An anchor at the end of the polynucleotide chain prevents it from passing completely through the pore onto the other side. For the return journey the potential is reversed, so that the process of unfolding now begins at the narrow end of the pore, and at this point the analysis is initiated.

"In contrast to the situation during forward translocation, no significant interactions appear to take place during the reverse trip," says Simmel.

On the basis of their experimental measurements, the researchers went on to construct a theoretical model that enabled them to predict the translocation dynamics of various hairpin structures with the aid of thermodynamic calculations of so-called "free-energy landscapes".

"This model could in the future provide the foundation of a procedure for the elucidation of the secondary structures of complex polynucleotides," says Gerland.

Explore further: Chemical biologists find new halogenation enzyme

More information: "Quantitative Analysis of the Nanopore Translocation Dynamics of Simple Structured Polynucleotides" S. Schink, S. Renner, K. Alim, V. Arnaut, F.C. Simmel, U. Gerland Biophysical Journal Vol. 102, January 2012, pp 1-11. doi: 10.1016/j.bpj.2011.11.4011

add to favorites email to friend print save as pdf

Related Stories

Moving polymers through pores

Jul 14, 2010

The movement of long chain polymers through nanopores is a key part of many biological processes, including the transport of RNA, DNA, and proteins. New research reported in The Journal of Chemical Physics, which ...

DNA through graphene nanopores

Jul 12, 2010

A team of researchers from Delft University of Technology (The Netherlands) announces a new type of nanopore devices that may significantly impact the way we screen DNA molecules, for example to read off their sequence. In ...

Recommended for you

Chemical biologists find new halogenation enzyme

Sep 15, 2014

Molecules containing carbon-halogen bonds are produced naturally across all kingdoms of life and constitute a large family of natural products with a broad range of biological activities. The presence of halogen substituents ...

Protein secrets of Ebola virus

Sep 15, 2014

The current Ebola virus outbreak in West Africa, which has claimed more than 2000 lives, has highlighted the need for a deeper understanding of the molecular biology of the virus that could be critical in ...

Protein courtship revealed through chemist's lens

Sep 15, 2014

Staying clear of diseases requires that the proteins in our cells cooperate with one another. But, it has been a well-guarded secret how tens of thousands of different proteins find the correct dancing partners ...

Decoding 'sweet codes' that determine protein fates

Sep 15, 2014

We often experience difficulties in identifying the accurate shape of dynamic and fluctuating objects. This is especially the case in the nanoscale world of biomolecules. The research group lead by Professor Koichi Kato of ...

User comments : 0