ERASE-Seq: Leveraging replicate measurements to enhance ultralow frequency variant detection in NGS data.

The accurate detection of ultralow allele frequency variants in DNA samples is of interest in both research and medical settings, particularly in liquid biopsies where cancer mutational status is monitored from circulating DNA. Next-generation sequencing (NGS) technologies employing molecular barcoding have shown promise but significant sensitivity and specificity improvements are still needed to detect mutations in a majority of patients before the metastatic stage. To address this we present analytical validation data for ERASE-Seq (Elimination of Recurrent Artifacts and Stochastic Errors), a method for accurate and sensitive detection of ultralow frequency DNA variants in NGS data. ERASE-Seq differs from previous methods by creating a robust statistical framework to utilize technical replicates in conjunction with background error modeling, providing a 10 to 100-fold reduction in false positive rates compared to published molecular barcoding methods. ERASE-Seq was tested using spiked human DNA mixtures with clinically realistic DNA input quantities to detect SNVs and indels between 0.05% and 1% allele frequency, the range commonly found in liquid biopsy samples. Variants were detected with greater than 90% sensitivity and a false positive rate below 0.1 calls per 10,000 possible variants. The approach represents a significant performance improvement compared to molecular barcoding methods and does not require changing molecular reagents.

What controls open-pore and residual currents in the first sensing zone of alpha-hemolysin nanopore? Combined experimental and theoretical study.

The electrophoretic transport of single-stranded DNA through biological nanopores such as alpha-hemolysin (αHL) is a promising and cost-effective technology with the potential to revolutionize genomics. The rational design of pores with the controlled polymer translocation rates and high contrast between different nucleotides could improve significantly nanopore sequencing applications. Here, we apply a combination of theoretical and experimental methods in an attempt to elucidate several selective modifications in the pore which were proposed to be central for the effective discrimination between purines and pyrimidines. Our nanopore test set includes the wild type αHL and six mutants (E111N/M113X/K147N) in which the cross-section and chemical functionality of the first constriction zone of the pore are modified. Electrophysiological recordings were combined with all-atom Molecular Dynamics simulations (MD) and a recently developed Brownian Dynamics (BROMOC) protocol to investigate residual ion currents and pore-DNA interactions for two homo-polymers e.g. poly(dA)40 or poly(dC)40 blocking the pore. The calculated residual currents and contrast in the poly(dA)40/poly(dC)40 blocked pore are in qualitative agreement with the experimental recordings. We showed that a simple structural metric allows rationalization of key elements in the emergent contrast between purines and pyrimidines in the modified αHL mutants. The shape of the pore and its capacity for hydrogen bonding to a translocated polynucleotide are two essential parameters for contrast optimization. To further probe the impact of these two factors in the ssDNA sensing, we eliminated the effect of the primary constriction using serine substitutions (i.e. E111S/M113S/T145S/K147S) and increased the hydrophobic volume of the central residue in the secondary constriction (L135I). This pore modification sharply increased the contrast between Adenine (A) and Cytosine (C).

Creating a Single Sensing Zone within an Alpha-Hemolysin Pore Via Site Directed Mutagenesis.

Although significant progress has recently been made towards realizing the goal of direct nanopore based DNA sequencing [1], there are still numerous hurdles that need to be overcome. One such hurdle associated with the use of the biological nanopore α-hemolysin (αHL) is the fact that the wild type channel contains three very distinct recognition or sensing regions within the ß-barrel [2, 3], making identification of the bases residing within or moving through the pore very difficult. Through site directed mutagenesis, we have been able to selectively remove one of two sensing regions while simultaneously enhancing the third. Our approach has led to the creation of αHL pores containing single sensing zones and provides the basis for engineering αHL pores suitable for direct DNA sequencing.

Bilayer-mediated clustering and functional interaction of MscL channels.

Mechanosensitive channels allow bacteria to respond to osmotic stress by opening a nanometer-sized pore in the cellular membrane. Although the underlying mechanism has been thoroughly studied on the basis of individual channels, the behavior of channel ensembles has yet to be elucidated. This work reveals that mechanosensitive channels of large conductance (MscL) exhibit a tendency to spatially cluster, and demonstrates the functional relevance of clustering. We evaluated the spatial distribution of channels in a lipid bilayer using patch-clamp electrophysiology, fluorescence and atomic force microscopy, and neutron scattering and reflection techniques, coupled with mathematical modeling of the mechanics of a membrane crowded with proteins. The results indicate that MscL forms clusters under a wide range of conditions. MscL is closely packed within each cluster but is still active and mechanosensitive. However, the channel activity is modulated by the presence of neighboring proteins, indicating membrane-mediated protein-protein interactions. Collectively, these results suggest that MscL self-assembly into channel clusters plays an osmoregulatory functional role in the membrane.

An improved open-channel structure of MscL determined from FRET confocal microscopy and simulation.

Mechanosensitive channels act as molecular transducers of mechanical force exerted on the membrane of living cells by opening in response to membrane bilayer deformations occurring in physiological processes such as touch, hearing, blood pressure regulation, and osmoregulation. Here, we determine the likely structure of the open state of the mechanosensitive channel of large conductance using a combination of patch clamp, fluorescence resonance energy transfer (FRET) spectroscopy, data from previous electron paramagnetic resonance experiments, and molecular and Brownian dynamics simulations. We show that structural rearrangements of the protein can be measured in similar conditions as patch clamp recordings while controlling the state of the pore in its natural lipid environment by modifying the lateral pressure distribution via the lipid bilayer. Transition to the open state is less dramatic than previously proposed, while the N terminus remains anchored at the surface of the membrane where it can either guide the tilt of or directly translate membrane tension to the conformation of the pore-lining helix. Combining FRET data obtained in physiological conditions with simulations is likely to be of great value for studying conformational changes in a range of multimeric membrane proteins.

Studying mechanosensitive ion channels using liposomes.

Mechanosensitive (MS) ion channels are the primary molecular transducers of mechanical force into electrical and/or chemical intracellular signals in living cells. They have been implicated in innumerable mechanosensory physiological processes including touch and pain sensation, hearing, blood pressure control, micturition, cell volume regulation, tissue growth, or cellular turgor control. Much of what we know about the basic physical principles underlying the conversion of mechanical force acting upon membranes of living cells into conformational changes of MS channels comes from studies of MS channels reconstituted into artificial liposomes. Using bacterial MS channels as a model, we have shown by reconstituting these channels into liposomes that there is a close relationship between the physico-chemical properties of the lipid bilayer and structural dynamics bringing about the function of these channels.

Rapid and improved reconstitution of bacterial mechanosensitive ion channel proteins MscS and MscL into liposomes using a modified sucrose method.

The bacterial mechanosensitive (MS) channels of small (MscS) and large (MscL) conductance have functionally been reconstituted into giant unilamellar liposomes (GUVs) using an improved reconstitution method in the presence of sucrose. This method gives significant time savings (preparation times as little as 6h) compared to the classical method of protein reconstitution which uses a dehydration/rehydration (D/R) procedure (minimum 2 days preparation time). Moreover, it represents the first highly reproducible method for functional reconstitution of MscS as well as MscS/MscL co-reconstitution. This novel procedure has the potential to be used for studies of other ion channels by liposome reconstitution.

Substrates induce conformational changes in human anion exchanger 1 (hAE1) as observed by fluorescence resonance energy transfer.

The one-for-one exchange of Cl(-) and HCO(3)(-) ions is catalyzed by human erythrocyte anion exchanger 1 (hAE1) through a ping-pong mechanism whereby the protein exists in two main conformations, with the single anion-binding site exposed at either the cytoplasmic (inner) side (E(i)) or the extracellular side (E(o)), with interconversion between the two states being possible only after anion binding. Steady-state and time-resolved resonance energy transfer (FRET) techniques were used to determine the distance of the binding site for diTBA (bis-(1,3-diethylthiobarbituric acid)trimethine oxonol), a high affinity fluorescent oxonol inhibitor of hAE1, from a benchmark site (probably Lys-430) labeled by external fluorescein maleimide (FM). Using red cell ghost membranes, energy transfer distances were measured in media containing different anions between FM as the donor, covalently attached to one monomer, and diTBA as the acceptor, reversibly bound to the adjacent monomer of a hAE1 dimer. Energy transfer increased significantly in chloride or bicarbonate buffers relative to conditions where no transportable anions were present, that is, in citrate buffer. These differences in transfer efficiencies were interpreted in light of the conformational distributions of hAE1 in various buffers and the possible effects of diTBA itself on the distribution. The analysis indicates that the diTBA binding site comes closer to the FM site by approximately 7 A in chloride buffer as compared to that in citrate (or equivalent changes in diTBA orientation occur) because of the effects of anion binding. This provides the first direct physical evidence for structural changes in hAE1 induced by substrates.

Conformational changes in the cytoplasmic domain of human anion exchanger 1 revealed by luminescence resonance energy transfer.

The cytoplasmic domain of the human erythrocyte anion exchanger 1 (cdAE1) serves as a center of organization for the red blood cell cytoskeleton as well as several metabolic enzymes and hemoglobin. The protein is known to undergo a reversible pH-dependent conformational change characterized by a 2-fold change in the intrinsic fluorescence and an 11 A change in the Stokes radius. While the exact changes in the molecular structure are unknown, on the basis of the crystal structure of the protein at pH 4.8 and site-directed mutagenesis studies, Zhou and Low (19) have proposed that the peripheral protein binding (PPB) domain of cdAE1 moves away from the dimerization domain in response to increasing alkalinity. To test this hypothesis, we have applied luminescence resonance energy transfer (LRET) to measure the intermonomer distance between donor and acceptor probes at the Cys201 site (located in the PPB domain) within the cdAE1 dimer. This distance was found to increase as the pH is increased from 5 to 10, in recombinant forms of both the wild type and a mutant (C317S) of cdAE1. Furthermore, LRET measurements in red blood cell inside-out vesicles indicate that when cdAE1 is linked to the membrane, the intermonomer distance is larger at pH 5, compared to that of the purified cdAE1 segments, and exhibits a different pH-dependent behavior. An increase in the distance was also observed on binding of a metabolic enzyme, glyceraldehyde-3-phosphate dehydrogenase, to cdAE1. These data provide the first demonstration of a defined change in the molecular structure of cdAE1, and also indicate that the structure under physiological conditions is different from the crystal structure determined at low pH.

A novel immobilization method for single protein spFRET studies.

We have developed a new method for immobilization of single proteins by utilizing streptavidin-biotin and protein L-antibody interactions on glass coverslips coated with polyethylene glycol. The method is particularly well suited for single-molecule fluorescence studies. A monomeric, detergent-solubilized bacterial transport protein, GlpT, and the dimeric cytoplasmic region of a mammalian transporter, cdAE1, were immobilized by our method with a high degree of specificity. The fluorescence from single molecules attached to the immobilized proteins was detected with a high signal/noise ratio. Single-pair fluorescence resonance energy transfer (spFRET) measurements on cdAE1 dimers indicate that the structure of the protein is not compromised and provide evidence that the cdAE1 protein can exist in at least two conformations under physiological conditions.

Use of luminescence resonance energy transfer to measure distances in the AE1 anion exchange protein dimer.

To understand how red blood cell and other proteins carry out their functions, it is necessary not only to have high-resolution crystal structures, but also to have methods that can measure changes in position of parts of the protein on the scale of Angstroms. The method of luminescence resonance energy transfer (LRET) has considerable advantages for this purpose, particularly for proteins, such as the AE1 anion exchange protein in the red cell, that are homodimers. We have applied this method, using a terbium maleimide chelate (TbM) as donor and fluorescein maleimide (FM) as acceptor, to measure the distance between the C201 residues in adjacent dimerized cytoplasmic domains of AE1 (cdAE1). The distance measured by LRET (40.8 A) corresponds closely with that calculated from the crystal structure of the cdAE1, indicating that the method can provide useful information for testing hypotheses concerning motions in this and other blood cell proteins.