Professor Meni Wanunu of Northeastern University delivered a lecture last Friday at the Dartmouth Physics Department on a developing field in biophysics: the analysis of DNA using nanopores. In the technique, single molecules of nucleic acids (~2.2 nm in diameter) are guided through pores as small as two or three nanometers in diameter by electric current. As the molecule passes through the pore, it alters the ionic current across the nanopore membrane, allowing for its detection. Nanopore membranes as thin as five or six nanometers, approximately the width of a cell’s membrane, allow for amplified signal from small fragments of nucleic acids to be detected. Molecules can be differentiated by the amplitude of the current anomaly they produce or by signal duration, reflective of differences in cross-sectional area and length of molecules.
Wanunu focused his presentation on the applications of nanopore nucleic acid detection. While nanopores are capable of sequencing DNA, they have yet to be optimized for the process due to the difficulty of resolving single base pairs. On the other hand, nanopores do not require prior amplification of the nucleic acids they read, permitting the counting of molecules. Wanunu described a potential application of nanopores in detecting microRNAs (miRNA), which are known to regulate gene expression. As miRNA molecules are present in low native abundances, detection methods must be able to discriminate between more common nucleic acids and the occasional regulatory elements. Using an unusual viral protein that binds double stranded RNA strands of defined length (21-23 base pairs, approximately that of miRNA), Wanunu’s lab was able to determine the concentration of specific miRNA molecules by binding probes to them, complexing the resulting double stranded probe:miRNA with the viral protein and counting the complexes by micropore analysis.
Further applications of nanopore analysis in extracting epigenetic information are found in the detection of chemical modifications to base pairs. In mammalian cells, a common modification is hydroxymethylation of cytosine bases, which is practically impossible to detect by conventional methods like gel electrophoresis and demands lengthy immunoprecipitation protocols. Although the physiological role of hydroxymethylated bases is not understood, it is possible that they are involved in complex epigenetic regulation of gene expression. Wanunu’s lab recently found that nanopore technology could distinguish hydroxymethylated bases from their methylated or unmethylated counterparts. Although the technique has yet to be fully optimized, nanopores can theoretically be used to detect hydroxymethylation in DNA sequences with as few as 1% of cytosines chemically modified in this way.
Wanunu believes that nanopore technology is a potential alternative to amplification-based analysis methods. Amplification of siRNA using reverse transcriptase PCR can lead to errors in product due to the difficulty of amplifying non-linear secondary structures in siRNA. Furthermore, technical refinements in reading signals from nanopores are breaking down barriers in resolution. The use of complementary metal-oxide semiconductor preamplifiers, for example, allows for the consistent detection of electrical signals at a bandwidth of 1 MHz, resolving events in the nanopore occurring in time intervals of one microsecond. Using such cutting edge nanopore technology in conjunction with other techniques of analysis allows for the detection of both genomic and epigenomic variation, a powerful tool for understanding the bases of gene expression.
Note: A recording of Professor Meni Wanunu’s presentation will be made available on the Dartmouth Physics website
http://www.dartmouth.edu/~physics/news/colloquia.archives.html (not uploaded at time of press)