Alternanthera yellow vein virus (AYVV); a betasatellite independent begomovirus infecting Sonchus palustris in Pakistan.

Satellites associated begomoviruses are the most diverse group of plant viruses in tropical and subtropical regions. In Pakistan, during field surveys in 2019-2020, Sonchus palustris (a weed plant) was observed showing begomovirus symptoms i.e., vein yellowing and mosaic patterns on leaves. Rolling circle amplification from total isolated DNA of symptomatic leaves was performed to amplify circular viral genomes. Subsequent cloning and sequencing showed that a new strain of Alternanthera yellow vein virus (AlYVV) is associated with vein yellowing disease of S. palustris. The identity percentage analysis through BLAST search and SDT analysis showed that the new strain is 94-98% identical to AlYVV isolates reported from Pakistan, India and China. In phylogenetic tree, it clustered with AlYVV-[PK:E prostrata:15-KX710155], AlYVV-[PK:E prostrata:13]-KX906697] and AlYVV-[PK:E prostrata:11]-KX906694] previously reported from Pakistan. There was no detectable level of betasatellite or any other satellite molecule in the samples studied here. Phylogenetic analysis of Rep and CP genes of AlYVV with corresponding genes of closely related viruses circulating in Southeast Asia showed intra-specific recombination involving both complementary and virion sense region of virus. Relaxed clock and Bayesian Skyline Plot analysis based on CP gene sequences indicated slight higher substitution rates (4.75 x 10-3 substitutions/nucleotide/year). In the Indian subcontinent satellite-associated monopartite begomoviruses predominately infect crops and non-crop plants. But AlYVV is found infecting mostly non-crop plants independent of satellite molecules. We hypothesize here that AlYVV evolved as a true monopartite begomovirus in the Indian sub-continent and could be a great threat to introduced crops under suitable conditions. Such studies are crucial to understand probable future epidemics of begomoviruses in the region.

Loss of the proprioception and touch sensation channel PIEZO2 in siblings with a progressive form of contractures.

Dominant mutations in PIEZO2, which codes for the principal mechanotransduction channel for proprioception and touch sensation, have been found to cause different forms of distal arthrogryposis. Some observations suggest that these dominant mutations induce a gain-of-function effect on the channel. Here, we report a consanguineous family with three siblings who showed short stature, scoliosis, gross motor impairment, and a progressive form of contractures involving the distal joints that is distinct from that found in patients with dominant mutations in PIEZO2. These siblings also displayed deficits in proprioception and touch sensation. Whole-exome sequencing performed in the three affected siblings revealed the presence of a rare homozygous variant (c.2708C>G; p.S903*) in PIEZO2. This variant is predicted to disrupt PIEZO2 function by abolishing the pore domain. Sanger sequencing confirmed that all three siblings are homozygous whereas their parents and an unaffected sibling are heterozygous for this variant. Recessive mutations in PIEZO2 thus appear to cause a progressive phenotype that overlaps with, while being mostly distinct from that associated with dominant mutations in the same gene.

Nanopore analysis of wild-type and mutant prion protein (PrP(C)): single molecule discrimination and PrP(C) kinetics.

Prion diseases are fatal neurodegenerative diseases associated with the conversion of cellular prion protein (PrP(C)) in the central nervous system into the infectious isoform (PrP(Sc)). The mechanics of conversion are almost entirely unknown, with understanding stymied by the lack of an atomic-level structure for PrP(Sc). A number of pathogenic PrP(C) mutants exist that are characterized by an increased propensity for conversion into PrP(Sc) and that differ from wild-type by only a single amino-acid point mutation in their primary structure. These mutations are known to perturb the stability and conformational dynamics of the protein. Understanding of how this occurs may provide insight into the mechanism of PrP(C) conversion. In this work we sought to explore wild-type and pathogenic mutant prion protein structure and dynamics by analysis of the current fluctuations through an organic α-hemolysin nanometer-scale pore (nanopore) in which a single prion protein has been captured electrophoretically. In doing this, we find that wild-type and D178N mutant PrP(C), (a PrP(C) mutant associated with both Fatal Familial Insomnia and Creutzfeldt-Jakob disease), exhibit easily distinguishable current signatures and kinetics inside the pore and we further demonstrate, with the use of Hidden Markov Model signal processing, accurate discrimination between these two proteins at the single molecule level based on the kinetics of a single PrP(C) capture event. Moreover, we present a four-state model to describe wild-type PrP(C) kinetics in the pore as a first step in our investigation on characterizing the differences in kinetics and conformational dynamics between wild-type and D178N mutant PrP(C). These results demonstrate the potential of nanopore analysis for highly sensitive, real-time protein and small molecule detection based on single molecule kinetics inside a nanopore, and show the utility of this technique as an assay to probe differences in stability between wild-type and mutant prion proteins at the single molecule level.

Direct observation of translocation in individual DNA polymerase complexes.

Complexes of phi29 DNA polymerase and DNA fluctuate on the millisecond time scale between two ionic current amplitude states when captured atop the α-hemolysin nanopore in an applied field. The lower amplitude state is stabilized by complementary dNTP and thus corresponds to complexes in the post-translocation state. We have demonstrated that in the upper amplitude state, the DNA is displaced by a distance of one nucleotide from the post-translocation state. We propose that the upper amplitude state corresponds to complexes in the pre-translocation state. Force exerted on the template strand biases the complexes toward the pre-translocation state. Based on the results of voltage and dNTP titrations, we concluded through mathematical modeling that complementary dNTP binds only to the post-translocation state, and we estimated the binding affinity. The equilibrium between the two states is influenced by active site-proximal DNA sequences. Consistent with the assignment of the upper amplitude state as the pre-translocation state, a DNA substrate that favors the pre-translocation state in complexes on the nanopore is a superior substrate in bulk phase for pyrophosphorolysis. There is also a correlation between DNA sequences that bias complexes toward the pre-translocation state and the rate of exonucleolysis in bulk phase, suggesting that during DNA synthesis the pathway for transfer of the primer strand from the polymerase to exonuclease active site initiates in the pre-translocation state.

Long dwell-time passage of DNA through nanometer-scale pores: kinetics and sequence dependence of motion.

A detailed understanding of the kinetics of DNA motion though nanometer-scale pores is important for the successful development of many of the proposed next-generation rapid DNA sequencing and analysis methods. Many of these approaches require DNA motion through nanopores to be slowed by several orders of magnitude from its native translocation velocity so that the translocation times for individual nucleotides fall within practical timescales for detection. With the increased dwell time of DNA in the pore, DNA-pore interactions begin to play an increasingly important role in translocation kinetics. In previous work, we and others observed that when the DNA dwell time in the pore is substantial (>1 ms), DNA motion in α-hemolysin (α-HL) pores leads to nonexponential kinetics in the escape of DNA out of the pore. Here we show that a three-state model for DNA escape, involving stochastic binding interactions of DNA with the pore, accurately reproduces the experimental data. In addition, we investigate the sequence dependence of the DNA escape process and show that the interaction strength of adenine with α-HL is substantially lower relative to cytosine. Our results indicate a difference in the process by which DNA moves through an α-HL nanopore when the motion is fast (microsecond timescale) as compared with when it is slow (millisecond timescale) and strongly influenced by DNA-pore interactions of the kind reported here. We also show the ability of wild-type α-HL to detect and distinguish between 5-methylcytosine and cytosine based on differences in the absolute ionic current through the pore in the presence of these two nucleotides. The results we present here regarding sequence-dependent (and dwell-time-dependent) DNA-pore interaction kinetics will have important implications for the design of methods for DNA analysis through reduced-velocity motion in nanopores.

Single-molecule bonds characterized by solid-state nanopore force spectroscopy.

Weak molecular interactions drive processes at the core of living systems, such as enzyme-substrate interactions, receptor-ligand binding, and nucleic acid replication. Single-molecule force spectroscopy is a remarkable tool for revealing molecular scale energy landscapes of noncovalent bonds, by exerting a mechanical force directly on an individual molecular complex and tracking its survival as a function of time and applied force. In principle, force spectroscopy methods can also be used for highly specific molecular recognition assays, by directly characterizing the strength of bonds between probe and target molecules. However, complexity and low throughput of conventional force spectroscopy techniques render such biosensing applications impractical. Here we demonstrate a straightforward single-molecule approach, suitable for both biophysical studies and molecular recognition assays, in which a approximately 3 nm silicon nitride nanopore is used to determine the bond lifetime spectrum of the biotin-neutravidin complex. Thousands of individual molecular complexes are captured and dissociated in the solid-state nanopore under constant applied forces, ranging from 400 to 900 mV, allowing us to extract the location of the energy barrier that governs the interaction, mapped at Deltax approximately 0.5 nm. These results highlight the capacity of a solid-state nanopore to detect and characterize intermolecular interactions and demonstrate how this could be applied to rapid, highly specific molecular detection assays.

Forming an alpha-hemolysin nanopore for single-molecule analysis.

Nanopore analysis of single molecules can be performed by measuring the modulation in ionic current passing through the nanopore while an individual biomolecule such as DNA or RNA is resident in, translocating through, or otherwise interacting with the pore. The corresponding current signature has been shown to reveal properties of the biomolecule and information on its interactions with the pore. The alpha-hemolysin nanopore remains the pore of choice, particularly for single-molecule analysis of nucleic acids, because of its internal dimensions, hydrophilicity, and low-noise characteristics. In this chapter we present a detailed protocol for forming a robust alpha-hemolysin nanopore (or multiple nanopores) for single-molecule analysis.

Nanopore force spectroscopy on DNA duplexes.

Force spectroscopy can be applied using nanopores to study charged molecules such as nucleic acids. This technique can be used to study the binding energy of a DNA duplex by threading an anchored single-stranded DNA (ssDNA) probe molecule through a nanopore (having a diameter large enough to accommodate only a single strand) and allowing target DNA on the backside of the pore to hybridize to the probe. Electric potential can be used to apply a force to the charged ssDNA in a direction tending to translocate the duplex through the pore. If the pore is only large enough to accept ssDNA, the duplex must dissociate for the probe to escape the pore. The dissociation time of the duplex can therefore be measured under applied force, and (provided that enough dissociation events have been recorded) a characteristic time scale for dissociation can be determined. In this chapter, we present a detailed protocol for performing nanopore force spectroscopy on DNA duplexes using one or more alpha-hemolysin nanopores. We present the details of the measurement of the duplex survival probability under force, and show that dissociation time scales for duplexes that are perfectly complimentary differ by greater than approximately two orders of magnitude from those containing a single sequence mismatch, offering opportunities for sequence detection.

Nonexponential kinetics of DNA escape from alpha-hemolysin nanopores.

Throughput and resolution of DNA sequence detection technologies employing nanometer scale pores hinge on accurate kinetic descriptions of DNA motion in nanopores. We present the first detailed experimental study of DNA escape kinetics from alpha-hemolysin nanopores and show that anomalously long escape times for some events result in nonexponential kinetics. From the distribution of first-passage times, we determine that the energy barrier to escape follows a Poisson-like distribution, most likely due to stochastic weak binding events between the DNA and amino acid residues in the pore.

Two dicot-infecting mastreviruses (family Geminiviridae) occur in Pakistan.

Most mastreviruses (family Geminiviridae) infect monocotyledonous hosts and are transmitted by leafhopper vectors. Only two mastrevirus species, Tobacco yellow dwarf virus from Australia and Bean yellow dwarf virus (BeYDV) from South Africa, have been identified whose members infect dicotyledonous plants. We have identified two distinct mastreviruses in chickpea stunt disease (CSD)-affected chickpea originating from Pakistan. The first is an isolate of BeYDV, previously only known to occur in South Africa. The second is a member of a new species with the BeYDV isolates as its closest relatives. A PCR-based diagnostic test was developed to differentiate these two virus species. Our results show that BeYDV plays no role in the etiology of CSD in Pakistan, while the second virus occurs widely in chickpea across Pakistan. A genomic clone of the new virus was infectious to chickpea (Cicer arietinum L.) and induced symptoms typical of CSD. We propose the use of the name Chickpea chlorotic dwarf Pakistan virus for the new species. The significance of these findings with respect to our understanding of the evolution, origin and geographic spread of dicot-infecting mastreviruses is discussed.