Detection of DNA methylation with a synthetic Nanopore
What is DNA methylation?
Methylation of cytosine is a covalent modification of DNA, in which the H5 of cytosine is replaced by a methyl group. In mammals, 60%-90% of all CpGs are methylated. Methylation adds information not encoded in the DNA sequence, but it does not interfere with the Watson-Crick pairing - the methyl group is positioned in the major groove of the DNA. The pattern of methylation controls protein binding to target sites on DNA, affecting changes in gene expression and in chromatin organization, often silencing genes, which physiologically orchestrates processes like differentiation, and pathologically leads to cancer [reference see publication below].
 Epigenetic code (image from Nature 441:143 (2006)) |
 DNA Methylation (image from scq.ubc.ca) |
Biological functions of DNA methylation
- Transcriptional gene silencing
- Genome stability
- Chromatin compaction
- Genome defense
- Suppression of homologous recombination between repeats
- X chromosome inactivation (females)
- (Nature Genetics 38:1359 (2006))
 Spot the Difference: Same Genes but a Different Kink in the Tail (Photograph courtesy of Emma Whitelaw, University of Sydney, Australia.) PLoS Biol 1:3 (2003)) |
 Hypermethylation of CpGs leads to cancer (Image from ncc.go.jp) |
Using nanopore to detect DNA methylation
System setup
we have reported [reference see publication below] measurements and simulations of the permeation of methylated DNA through a synthetic nanopore, using an electric field to force single molecules to translocate across the membrane through the pore (see also the our nanopore site). Two fragments of genomic DNA studied are known to control expression based on methylation status: MS3 and BRCA1. MS3 is one of the CTCF binding sites of the Igf2 imprinting control region (ICR). Methylation of MS3 prevents CTCF binding, allowing an enhancer to reach Igf2 and turn on expression. Aberrant hypermethylation causes elevated expression of Igf2, which has been shown to promote cancer [reference see publication below]. In contrast, BRCA1 is a tumor suppressor gene used to repair DNA. Methylation of the BRCA1 promoter causes binding of a protein (MeCP2) that inhibits expression leading to mutations and breast/ovarian cancer [reference see publication below]. We investigated the permeability of MS3 and BRCA1 with different methylation levels and profiles through two pores with similar (~1.8nm) diameters. The patterns of methylated CpG sites in the five strands used here are shown in Figure below.
Patterns of methylated CpG sites
Experimental results
We observe a voltage threshold for permeation of methylated DNA through a < 2nm diameter pore, which we attribute to the stretching transition of DNA, that can differ by >1V/20nm depending on the methylation level, but does not depend sensitively on DNA sequence. The threshold for unmethylated MS3 is about 3.6 V while hemi- and fully-methylated MS3 show a threshold of 3.2 V and 2.7 V respectively. Though the threshold is appartently related to the methylation level, it is relatively insensitive to the DNA sequence, as evident from the comparison between the permeation of MS3 and BRCA1 through the 1.7±0.2nm pore shown below. The BRCA1 and MS3 sequences are different, but the thresholds for stretching are close: 3.6 V and 3.8 V respectively. Yet, fully methylated BRCA1, which has 12 methylated CpG sites, and fully methylated MS3, which has a comparable number, both show a similar shift in threshold upon methylation to 2.7 V and 2.5 V, respectively.
Simulation results
At 4V bias, we recognized a significant difference between the translocation speeds of methylated and unmethylated DNA, namely, speed of 1.0 nm/ns and 0.8 nm/ns, respectively. The velocity difference implies that methylated DNA passes the pore more readily than unmethylated DNA, which indeed is consistent with the threshold voltage difference measured. Conformations of the DNAs are shown in the figure below. One can recognize that both translocating DNAs exhibit a B-form structure. Howevre, methylated DNA is considerably more ordered and adheres more closely to the ideal B-form than does unmethylated DNA. This behavior is also evident from the root means square fluctuation (RMSF) values of the two molecules (central segment only), with methlated DNA under 4 V voltage having an RMSF value of 2.9 Angstrom, while unmethylated DNA has an RMSF value of 4.9 Angstrom. More detail can be found here.
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Publications
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Publications Database Nanoelectromechanics of Methylated DNA in a Synthetic Nanopore U. Mirsaidov, W. Timp, X. Zou, V. Dimitrov, K. Schulten, A. P. Feinberg, and G. Timp Biophysical Journal, in press
Investigators
Simulations ExperimentRelated TCB Group Projects
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