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Nucleotide discrimination with DNA immobilized in the MspA nanopore
Elizabeth A Manrao1, Ian M Derrington, Mikhail Pavlenok
1Department of Physics, University of Washington, Seattle, Washington, United States of America.
Abstract:
Nanopore sequencing has the potential to become a fast and low-cost DNA sequencing platform. An ionic current passing through a small pore would directly map the sequence of single stranded DNA (ssDNA) driven through the constriction. The pore protein, MspA, derived from Mycobacterium smegmatis, has a short and narrow channel constriction ideally suited for nanopore sequencing. To study MspA's ability to resolve nucleotides, we held ssDNA within the pore using a biotin-NeutrAvidin complex. We show that homopolymers of adenine, cytosine, thymine, and guanine in MspA exhibit much larger current differences than in α-hemolysin. Additionally, methylated cytosine is distinguishable from unmethylated cytosine. We establish that single nucleotide substitutions within homopolymer ssDNA can be detected when held in MspA's constriction. Using genomic single nucleotide polymorphisms, we demonstrate that single nucleotides within random DNA can be identified. Our results indicate that MspA has high signal-to-noise ratio and the single nucleotide sensitivity desired for nanopore sequencing devices.
Insights
The MspA protein from Mycobacterium smegmatis shows high signal-to-noise for nanopore sequencing. It can accurately detect single nucleotide differences in DNA, including methylated cytosine.
Area of Science:
- Biophysics
- Genomics
- Molecular Biology
Background:
- Nanopore sequencing offers a promising avenue for rapid and cost-effective DNA analysis.
- The ionic current through a nanopore can directly map single-stranded DNA (ssDNA) sequences.
- The Mycobacterium smegmatis protein A (MspA) possesses a narrow pore constriction suitable for nucleotide resolution.
Purpose of the Study:
- To evaluate the MspA protein's capability in distinguishing individual nucleotides within ssDNA.
- To assess MspA's potential for high-resolution nanopore sequencing applications.
Main Methods:
- Holding ssDNA within the MspA pore using a biotin-NeutrAvidin complex.
- Measuring ionic current changes as ssDNA passes through the MspA constriction.
- Analyzing current differences for homopolymers and single nucleotide substitutions.
Main Results:
- MspA demonstrated significantly larger current differences for DNA homopolymers compared to α-hemolysin.
- Distinguishing between methylated and unmethylated cytosine was achieved.
- Detection of single nucleotide substitutions in homopolymers and identification of single nucleotides in random DNA were successful.
- MspA exhibited high signal-to-noise ratio and single nucleotide sensitivity.
Conclusions:
- MspA is a highly sensitive and specific nanopore for DNA sequencing.
- MspA's properties make it a strong candidate for developing advanced nanopore sequencing technologies.
- The ability to detect DNA sequence variations, including epigenetic modifications, opens new possibilities in genomic research.
