Biology subjects

Wanunu, M.

Publications and source records attributed to Wanunu, M..

7 recordsLinked to original sources

bioRxiv2022

Messenger-RNA Modification Standards and Machine Learning Models Facilitate Absolute Site-Specific Pseudouridine Quantification

Enzyme-mediated chemical modifications to mRNA are important for fine-tuning gene expression, but they are challenging to quantify due to low copy number and limited tools for accurate detection. Existing studies have typically focused on the identification and impact of adenine modifications on mRNA (m6A and inosine) due to the availability of analytical methods. The pseudouridine ({Psi}) mRNA modification is also highly abundant but difficult to detect and quantify because there is no available antibody, it is mass silent, and maintains canonical basepairing with adenine. Nanopores may be used to directly identify {Psi} sites in RNAs using a systematically miscalled base, however, this approach is not quantitative and highly sequence dependent. In this work, we apply supervised machine learning models that are trained on sequence-specific, synthetic controls to endogenous transcriptome data and achieve the first quantitative {Psi} occupancy measurement in human mRNAs. Our supervised machine learning models reveal that for every site studied, different signal parameters are required to maximize {Psi} classification accuracy. We show that applying our model is critical for quantification, especially in low-abundance mRNAs. Our engine can be used to profile {Psi}-occupancy across cell types and cell states, thus providing critical insights about physiological relevance of {Psi} modification to mRNAs.

genomics↗

bioRxiv2021

Detection of pseudouridine modifications and type I/II hypermodifications in human mRNAs using direct, long-read sequencing

We developed and applied a semi-quantitative method for high-confidence identification of pseudouridylated sites on mammalian mRNAs via direct long-read nanopore sequencing. A comparative analysis of a modification-free transcriptome reveals that the depth of coverage and specific k-mer sequences are critical parameters for accurate basecalling. By adjusting these parameters for high-confidence U-to-C basecalling errors, we identified many known sites of pseudouridylation and uncovered new uridine-modified sites, many of which fall in k-mers that are known targets of pseudouridine synthases. Identified sites were validated using 1,000-mer synthetic RNA controls bearing a single pseudouridine in the center position which demonstrate systematical under-calling using our approach. We identify mRNAs with up to 7 unique modification sites. Our pipeline allows direct detection of low-, medium-, and high-occupancy pseudouridine modifications on native RNA molecules from nanopore sequencing data as well as multiple modifications on the same strand.

bioengineering↗

bioRxiv2021

Unidirectional Single-File Transport of Full-Length Proteins Through a Nanopore

Nanopore sensors could revolutionize single-molecule proteomics by providing a means for identification of known proteins through fingerprinting or by de novo sequencing. However, the complex chemical and physical properties of proteins present multiple challenges to the conventional nanopore sensing method, predominantly, the single-file threading of a protein chain into a nanopore and its transport through it. Herein we describe a general approach for realizing unidirectional transport of full-length proteins through nanopores. We show that the combination of a chemically resistant biological nanopore platform and a high concentration guanidinium chloride buffer enables protein unfolding and unidirectional transport through a pore, propelled by an electroosmotic effect that largely owes to the guanidinium chloride presence. The uniform and slow (~10 {micro}s/amino acid) single-file transport, when combined with supervised machine learning of the electrical current signatures obtained, allows us to use to discern the protein threading orientation and identity. In conjunction with a method for tail-modification of native proteins and higher-resolution nanopores, our approach could offer a path towards direct single-molecule protein fingerprinting without the requirement of a motor enzyme.

biophysics↗

bioRxiv2021

Control of subunit stoichiometry in single-chain MspA nanopores

Transmembrane protein channels enable fast and highly sensitive electrical detection of single molecules. Nanopore sequencing of DNA was achieved using an engineered Mycobacterium smegmatis porin A (MspA) in combination with a motor enzyme. Due to its favorable channel geometry, the octameric MspA pore exhibits the highest current level as compared to other pore proteins. To date, MspA is the only protein nanopore with a published record of DNA sequencing. While widely used in commercial devices, nanopore sequencing of DNA suffers from significant base-calling errors due to stochastic events of the complex DNA-motor-pore combination and the contribution of up to five nucleotides to the signal at each position. Asymmetric mutations within subunits of the channel protein offer an enormous potential to improve nucleotide resolution and sequencing accuracy. However, random subunit assembly does not allow control of the channel composition of MspA and other oligomeric protein pores. In this study, we showed that it is feasible to convert octameric MspA into a single-chain pore by connecting eight subunits using peptide linkers. We constructed single-chain MspA trimers, pentamers, hexamers and heptamers to demonstrate that it is feasible to alter the subunit stoichiometry and the MspA pore diameter. All single-chain MspA proteins formed functional channels in lipid bilayer experiments. Importantly, we demonstrated that single-chain MspA discriminated all four nucleotides identical to MspA produced from monomers. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications. STATEMENT OF SIGNFICANCENanopore sequencing of DNA is a fast and cheap technology that uniquely delivers multi-kilobase reads. It is currently used world-wide in many applications such as genome sequencing, epigenetics, and surveillance of viral and bacterial pathogens and has started to revolutionize human lives in medicine, agriculture and environmental studies. However, the high base-calling error rates prevent nanopore DNA sequencing from reaching its full potential. In this study, we converted octameric MspA into a single-chain pore enabling asymmetric mutations to fine-tune the pore geometry and chemistry and address the shortcomings of nanopores. Thus, single-chain MspA constitutes a new milestone in its development and adaptation as a biosensor for DNA sequencing and many other applications.

biophysics↗

bioRxiv2021

CMOS electrochemical imaging arrays for thedetection and classification of microorganisms

Microorganisms account for most of the biodiversity on earth. Yet while there are increasingly powerful tools for studying microbial genetic diversity, there are fewer tools for studying microorganisms in their natural environments. In this paper, we present recent advances in CMOS electrochemical imaging arrays for detecting and classifying microorganisms. These microscale sensing platforms can provide non-optical measurements of cell geometries, behaviors, and metabolic markers. We review integrated electronic sensors appropriate for monitoring microbial growth, and present measurements of single-celled algae using a CMOS sensor array with thousands of active pixels. Integrated electrochemical imaging can contribute to improved medical diagnostics and environmental monitoring, as well as discoveries of new microbial populations.

bioengineering↗

bioRxiv2021

Electrical Unfolding of Cytochrome c During Translocation Through a Nanopore Constriction

Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field-driven translocation behavior of cytochrome c (cyt c) through ultrathin silicon nitride (SiNx) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5 nm diameter pore we find that, in a threshold electric field regime of [~]30-100 MV/m, cyt c is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt c is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5 nm and 2.0 nm diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow ([~]ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens new avenues for exploring protein folding structures, internal contacts, and electric field-induced deformability. Significance StatementCan localized electric fields drive the complete unfolding of a protein molecule? Protein unfolding prior to its translocation through a nanopore constriction is an important step in protein transport across biological membranes and also an important step in nanopore-based protein sequencing. We studied here the electric-field-driven translocation behavior of a model protein (cyt c) through nanopores of diameters ranging from 1.5 to 5.5 nm. These single molecule measurements show that electric fields at the nanopore constriction can select both partially and fully unfolded protein conformations. Zero-field free energy gaps between these conformations, found using a simple thermodynamic model, are in remarkable agreement with previously reported studies of cyt c unfolding energetics.

biophysics↗

bioRxiv2020

Stable Polymer Bilayers for Protein Channel Recordings at High Guanidinium Chloride Concentrations

Use of chaotropic reagents is common in biophysical characterization of biomolecules. When the study involves transmembrane protein channels, the stability of the protein channel and supporting bilayer membrane must be considered. In this letter we show that planar bilayers composed of poly(1,2-butadiene)-b-poly(ethylene oxide) diblock copolymer are stable and leak-free at high guanidinium chloride concentrations, in contrast to diphytanoyl phosphatidylcholine bilayers which exhibit deleterious leakage under similar conditions. Further, insertion and functional analysis of channels such as -hemolysin and MspA are straightforward in these polymer membranes. Finally, we demonstrate that -hemolysin channels maintain their structural integrity at 2M guanidinium chloride concentrations using blunt DNA hairpins as molecular reporters.

biophysics↗