Single-molecule DNA detection with an engineered MspA protein nanopore

425

498

The sequencing of individual DNA strands with nanopores is under investigation as a rapid, low-cost platform in which bases are identified in order as the DNA strand is transported through a pore under an electrical potential. Although the preparation of solid-state nanopores is improving, biological nanopores, such as ␣-hemolysin (␣HL), are advantageous because they can be precisely manipulated by genetic modification. Here, we show that the transmembrane ␤-barrel of an engineered ␣HL pore contains 3 recognition sites that can be used to identify all 4 DNA bases in an immobilized single-stranded DNA molecule, whether they are located in an otherwise homopolymeric DNA strand or in a heteropolymeric strand. The additional steps required to enable nanopore DNA sequencing are outlined.␣-hemolysin ͉ DNA sequencing ͉ genomics ͉ protein engineering ͉ protein pore

mentioning

confidence: 65%

Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore

Stoddart

Heron

Mikhailova

et al. 2009

425

498

“…Single M2MspA channels were inserted in lipid bilayers in 0.3 M KCl, 10 mM HEPES/KOH (pH 8.00 ± 0.05) at 23°C as previously described (18,22). The channels used for this study had currents between 110 and 120 pA at 180 mV (trans-side positive) upon insertion.…”

Section: Methodsmentioning

confidence: 99%

“…The M2MspA protein (22) used to form single nanopores was prepared in our laboratory. Briefly, the ORF for M2MspA was constructed by overlapping PCR.…”

Section: Methodsmentioning

confidence: 99%

Error rates for nanopore discrimination among cytosine, methylcytosine, and hydroxymethylcytosine along individual DNA strands

Schreiber

Wescoe

Abu-Shumays

et al. 2013

174

156

Cytosine, 5-methylcytosine, and 5-hydroxymethylcytosine were identified during translocation of single DNA template strands through a modified Mycobacterium smegmatis porin A (M2MspA) nanopore under control of phi29 DNA polymerase. This identification was based on three consecutive ionic current states that correspond to passage of modified or unmodified CG dinucleotides and their immediate neighbors through the nanopore limiting aperture. To establish quality scores for these calls, we examined ∼3,300 translocation events for 48 distinct DNA constructs. Each experiment analyzed a mixture of cytosine-, 5-methylcytosine-, and 5-hydroxymethylcytosine-bearing DNA strands that contained a marker that independently established the correct cytosine methylation status at the target CG of each molecule tested. To calculate error rates for these calls, we established decision boundaries using a variety of machine-learning methods. These error rates depended upon the identity of the bases immediately 5′ and 3′ of the targeted CG dinucleotide, and ranged from 1.7% to 12.2% for a single-pass read. We estimate that Q40 values (0.01% error rates) for methylation status calls could be achieved by reading single molecules 5-19 times depending upon sequence context.MspA | epigenetics E pigenetic modifications of DNA help regulate gene transcription in biological cells. In mammals, 5-methylcytosine (mC) modification of CG dinucleotides is known to influence development (1, 2) and contribute to human diseases including cancer (3). Other modifications have been detected at carbon 5 of cytosine including 5-hydroxymethylcytosine (hmC) (4), and more recently 5-formylcytosine, and 5-carboxycytosine (5). Physiological roles for hmC in carcinogenesis and embryonic stem cell differentiation have been proposed (6).High-throughput techniques for mC detection are based on bisulfite treatment of genomic DNA (7). In the conventional assay, cytosine (but not mC nor hmC) is converted to uracil (8). Thus, positions not converted to uracil identify cytosines that were modified in the original genomic sequence. In a landmark paper, Lister et al. (9) used this technique to map genome-wide cytosine methylation in human embryonic stem cells and fetal lung fibroblasts at single-nucleotide precision. Recently, bisulfite strategies for discriminating between mC and hmC using the Tet1 enzyme (10) or by chemical modification of hmC (11) have been described.Single-molecule techniques have emerged as possible alternatives to bisulfite treatment for detecting epigenetic modifications of DNA (12). These single-molecule approaches share several useful features including few processing steps before sequence analysis, long reads that routinely exceed several thousand nucleotides, and the ability to read native DNA strands in heterogeneous mixtures. The most advanced of these single-molecule techniques, from Pacific Biosciences, uses fluorescence to detect labeled nucleotide triphosphates during daughter-strand elongation. This elongation is catalyzed by a DNA pol...

“…In addition to their biological interest they are increasingly relevant to biotechnology, serving as scaffolds for bacterial surface display (2,3) and atomically precise pores for nanopore-based DNA sequencing. Although the suitability of natural β-barrel membrane proteins for biotechnology has been improved by protein engineering (3)(4)(5)(6)(7)(8)(9)(10), the ability to design membrane proteins de novo would deliver tools customized to meet the demands of each application.…”

mentioning

confidence: 99%

Computational redesign of the lipid-facing surface of the outer membrane protein OmpA

Stapleton

Whitehead

Nanda

2015

Advances in computational design methods have made possible extensive engineering of soluble proteins, but designed β-barrel membrane proteins await improvements in our understanding of the sequence determinants of folding and stability. A subset of the amino acid residues of membrane proteins interact with the cell membrane, and the design rules that govern this lipid-facing surface are poorly understood. We applied a residue-level depth potential for β-barrel membrane proteins to the complete redesign of the lipid-facing surface of Escherichia coli OmpA. Initial designs failed to fold correctly, but reversion of a small number of mutations indicated by backcross experiments yielded designs with substitutions to up to 60% of the surface that did support folding and membrane insertion. T he β-barrel membrane proteins comprise one of the two structural classes of integral membrane proteins. They are found within the outer membranes of bacteria, mitochondria, and chloroplasts, where they perform a range of structural, transport, and catalytic functions (1). In addition to their biological interest they are increasingly relevant to biotechnology, serving as scaffolds for bacterial surface display (2, 3) and atomically precise pores for nanopore-based DNA sequencing. Although the suitability of natural β-barrel membrane proteins for biotechnology has been improved by protein engineering (3-10), the ability to design membrane proteins de novo would deliver tools customized to meet the demands of each application.De novo design provides a stringent test of our understanding of the determinants of protein folding and stability. Protein design software [e.g., Rosetta (11,12)] has made tremendous strides in addressing the design problem for small water-soluble proteins (13-15), and design of simplified model α-helical membrane proteins including single transmembrane helices and small bundles (16)(17)(18)(19)(20) has also been accomplished. In contrast, a designed β-barrel membrane protein has yet to be reported, perhaps as a consequence of the unique design challenges presented by the folding pathway and architecture of these proteins. Unlike the α-helical membrane proteins, nascent β-barrel membrane proteins must transit the periplasm to the outer membrane, where folding and membrane insertion are thought to occur in concert (21,22). An extensive network of chaperones maintains the solubility of the unfolded barrel and guides membrane insertion. The C-terminal β-strand is known to interact with the BAM chaperone complex (23-25), which assists the folding of all β-barrel membrane proteins. However, despite recent progress (26-30), we do not fully understand how interactions between chaperones and transiting membrane proteins are directed by sequence-encoded information.Further complicating design is the inside-out architecture of β-barrel membrane proteins. In place of a hydrophobic core is either a central water-filled pore or a solid core composed of polar side chains. The lipid bilayer becomes increasingly hydrophobic...