Repurposing an antimicrobial peptide for the development of a dual ion channel/molecular receptor-like platform for metal ion detection.

The presence of non-essential metals in the environment as contaminants is prone to cause hazardous health problems following accumulation in the human body and the ensuing toxic effects. This calls for continuous discovery and innovation in the realm of developing easy-to-operate, cheap and sensitive sensors. Herein, we describe the proof of concept approach for designing a molecular receptor-like, chimeric sensor based on the pore-forming peptide alamethicin (Alm), tethered via a linker with an ultrashort peptide nucleic acid (PNA) moiety, capable of generating functional ion channel oligomers in planar lipid membranes. The working principle of the sensor exploits the ability of Hg2+ ions to complex mismatching thymine-thymine sequences between the PNA receptor moiety on Alm oligomers and free, thymine-based, single-stranded DNAs (ssDNAs) in solution, thus creating a stable base pair at the oligomer entrance. This generates a transducing mechanism which converts the metal ion complexation into a specific electrical signature of the self-assembled Alm oligomers, enabling selective Hg2+ ion detection. The platform is programmable, whereby the simple exchange of the PNA sequence and its ssDNA counterpart in solution rendered the system selective for Cu2+ ion detection. With further optimization, the presented solution has the potential to translate into miniaturized, cost-effective biosensors suitable for the real-time, label-free and continuous detection of metal ions or other biomolecules.

Controlling DNA Fragments Translocation across Nanopores with the Synergic Use of Site-Directed Mutagenesis, pH-Dependent Charge Tuning, and Electroosmotic Flow.

Biological and solid-state nanopores are at the core of transformative techniques and nanodevices, democratizing the examination of matter and biochemical reactions at the single-molecule level, with low cost, portability, and simplicity in operation. One of the crucial hurdles in such endeavors is the fast analyte translocation, which limits characterization, and a rich number of strategies have been explored over the years to overcome this. Here, by site-directed mutagenesis on the α-hemolysin protein nanopore (α-HL), sought to replace selected amino acids with glycine, electrostatic binding sites were induced on the nanopore's vestibule and constriction region and achieved in the most favorable case a 20-fold increase in the translocation time of short single-stranded DNA (ssDNA) at neutral pH, with respect to the wild-type (WT) nanopore. We demonstrated an efficient tool of controlling the ssDNA translocation time, via the interplay between the nanopore-ssDNA surface electrostatic interactions and electroosmotic flow, all mediated by the pH-dependent ionization of amino acids lining the nanopore's translocation pathway. Our data also reveal the nonmonotonic, pH-induced alteration of ssDNA average translocation time. Unlike mildly acidic conditions (pH ∼ 4.7), at a pH ∼ 2.8 maintained symmetrically or asymmetrically across the WT α-HL, we evidenced the manifestation of a dominant electroosmotic flow, determining the speeding up of the ssDNA translocation across the nanopore by counteracting the ssDNA-nanopore attractive electrostatic interactions. We envision potential applications of the presented approach by enabling easy-to-use, real-time detection of short ssDNA sequences, without the need for complex biochemical modifications to the nanopore to mitigate the fast translocation of such sequences.

Correction: Considerable slowdown of short DNA fragment translocation across a protein nanopore using pH-induced generation of enthalpic traps inside the permeation pathway.

Correction for 'Considerable slowdown of short DNA fragment translocation across a protein nanopore using pH-induced generation of enthalpic traps inside the permeation pathway' by Loredana Mereuta et al., Nanoscale, 2023, 15, 14754-14763, https://doi.org/10.1039/D3NR03344A.

Considerable slowdown of short DNA fragment translocation across a protein nanopore using pH-induced generation of enthalpic traps inside the permeation pathway.

A pressing challenge in the realm of nanopore-based sensing technologies for nucleic acid characterization has been the cheap and efficient control of analyte translocation. To address this, a plethora of methods were tested, including mutagenesis, molecular motors, enzymes, or the optimization of experimental conditions. Herein, we present a paradigm exploiting the manipulation of electrostatic interactions between 22-mer single-stranded DNAs (22_ssDNA) and low pH-induced charges in the alpha-hemolysin (α-HL) nanopore, to efficiently control the passage of captured molecules. We discovered that in electrolytes buffered at pH = 5 and pH = 4.5 where the nanopore's vestibule and lumen become oppositely charged as compared to that at neutral pH, the electrostatic anchoring at these regions of a 22_ssDNA fragment leads to a dramatic increase of the translocation time, orders of magnitude larger compared to that at neutral pH. This pH-dependent tethering effect is reversible, side invariant, and sensitive to the ionic strength and ssDNA contour length. In the long run, our discovery has the potential to provide a simple read-out of the sequence of bases pertaining to short nucleotide sequences, thus extending the efficacy of current nanopore-based sequencers.

Synthetic Receptor Based on a Peptide Antibiotic-Functionalized Chimera for Hybridization-Based Polynucleotide Detection.

Nanopores offer highly sensitive, low-cost, and single-molecule sensing capabilities, and the societal impact of this approach is best captured by the advent of nanopore-based DNA detection and sequencing technologies, which extract genomic information without amplification. To address a critical difficulty plaguing such undertakings involving especially protein-based nanopores isolated in lipid bilayers, namely, the formation of a stable, long-lasting single nanopore, we pioneer herein an approach for generating functional nanostructures enabling small single-stranded DNA (ssDNA) detection. We designed a dynamic hybrid construct by appending extramembrane peptide nucleic acid (PNA) segments to the C-terminus of modified ion channel-forming alamethicin monomers. We found that the resulting chimeric molecules successfully coassemble in a voltage-dependent manner in planar lipid membranes generating diameter-variable oligomers. The subsequent interaction at the flexible extramembrane segment of such formed dynamic nanopores with aqueously added complementary ssDNA fragments leads to overall conformational alterations affecting the peptide assembly state kinetics and mediated ionic current. Such recognition events were found specific to the primary structure of target ssDNA and uninhibited the presence of serum. Our platform demonstrates the feasibility of designing an entirely new class of versatile chimeric biosensors, for which, dependent upon the nature of the attached receptor moiety and underlying recognition chemistry, the applicability area may extend to other analytes.

Probing the Hepatitis B Virus E-Antigen with a Nanopore Sensor Based on Collisional Events Analysis.

Real-time monitoring, simple operation, and cheaper methods for detecting immunological proteins hold the potential for a solid influence on proteomics and human biology, as they can promote the onset of timely diagnoses and adequate treatment protocols. In this work we present an exploratory study suggesting the applicability of resistive-pulse sensing technology in conjunction with the α-hemolysin (α-HL) protein nanopore, for the detection of the chronic hepatitis B virus (HBV) e-antigen (HBeAg). In this approach, the recognition between HBeAg and a purified monoclonal hepatitis B e antibody (Ab(HBeAg)) was detected via transient ionic current spikes generated by partial occlusions of the α-HL nanopore by protein aggregates electrophoretically driven toward the nanopore's vestibule entrance. Despite the steric hindrance precluding antigen, antibody, or antigen-antibody complex capture inside the nanopore, their stochastic bumping with the nanopore generated clear transient blockade events. The subsequent analysis suggested the detection of protein subpopulations in solution, rendering the approach a potentially valuable label-free platform for the sensitive, submicromolar-scale screening of HBeAg targets.

A Nanopore Sensor for Multiplexed Detection of Short Polynucleotides Based on Length-Variable, Poly-Arginine-Conjugated Peptide Nucleic Acids.

Real-time and easy-to-use detection of nucleic acids is crucial for many applications, including medical diagnostics, genetic screening, forensic science, or monitoring the onset and progression of various diseases. Herein, an exploratory single-molecule approach for multiplexed discrimination among similar-sized single-stranded DNAs (ssDNA) is presented. The underlying strategy combined (i) a method based on length-variable, short arginine (poly-Arg) tags appended to peptide nucleic acid (PNA) probes, designed to hybridize with selected regions from complementary ssDNA targets (cDNA) in solution and (ii) formation and subsequent detection with the α-hemolysin nanopore of (poly-Arg)-PNA-cDNA duplexes containing two overhangs associated with the poly-Arg tail and the non-hybridized segment from ssDNA. We discovered that the length-variable poly-Arg tail marked distinctly the molecular processes associated with the nanopore-mediated duplexes capture, trapping and unzipping. This enabled the detection of ssDNA targets via the signatures of (poly-Arg)-PNA-cDNA blockade events, rendered most efficient from the ß-barrel entrance of the nanopore, and scaled proportional in efficacy with a larger poly-Arg moiety. We illustrate the approach by sensing synthetic ssDNAs designed to emulate fragments from two regions of SARS-CoV-2 nucleocapsid phosphoprotein N-gene.

A Single-Molecule Insight into the Ionic Strength-dependent, Cationic Peptide Nucleic Acids-Oligonucleotides Interactions.

To alleviate solubility-related shortcomings associated with the use of neutral peptide nucleic acids (PNA), a powerful strategy is incorporate various charged sidechains onto the PNA structure. Here we employ a single-molecule technique and prove that the ionic current blockade signature of free poly(Arg)-PNAs and their corresponding duplexes with target ssDNAs interacting with a single α-hemolysin (α-HL) nanopore is highly ionic strength dependent, with high salt-containing electrolytes facilitating both capture and isolation of such complexes. Our data illustrate the effect of low ionic strength in reducing the effective volume of free poly(Arg)-PNAs and augmentation of their electrophoretic mobility while traversing the nanopore. We found that unlike in high salt electrolytes, the specific hybridization of cationic moiety-containing PNAs with complementary negatively charged ssDNAs in a salt concentration as low as 0.5 M is dramatically impeded. We suggest a scenario in which reduced charge screening by counterions in low salt electrolytes enables non-specific, electrostatic interactions with the anionic backbone of polynucleotides, thus reducing the ability of PNA-DNA complementary association via hydrogen bonding patterns. We applied an experimental strategy with spatially-separated poly(Arg)-PNAs and ssDNAs, and present evidence at the single-molecule level suggestive of the real-time, long-range interactions-driven formation of poly(Arg)-PNA-DNA complexes, as individual strands entering the nanopore from opposite directions collide inside a nanocavity.

Single-molecule, hybridization-based strategies for short nucleic acids detection and recognition with nanopores.

DNA nanotechnology has seen large developments over the last 30 years through the combination of detection and discovery of DNAs, and solid phase synthesis to increase the chemical functionalities on nucleic acids, leading to the emergence of novel and sophisticated in features, nucleic acids-based biopolymers. Arguably, nanopores developed for fast and direct detection of a large variety of molecules, are part of a revolutionary technological evolution which led to cheaper, smaller and considerably easier to use devices enabling DNA detection and sequencing at the single-molecule level. Through their versatility, the nanopore-based tools proved useful biomedicine, nanoscale chemistry, biology and physics, as well as other disciplines spanning materials science to ecology and anthropology. This mini-review discusses the progress of nanopore- and hybridization-based DNA detection, and explores a range of state-of-the-art applications afforded through the combination of certain synthetically-derived polymers mimicking nucleic acids and nanopores, for the single-molecule biophysics on short DNA structures.

Teaching an old dog new tricks: A lipid membrane-based electric immunosensor for real-time probing of the spike S1 protein subunit from SARS-CoV-2.

Fast, cheap, and easy to implement point-of-care testing for various pathogens constituted a game changer in past years due to its potential for early disease diagnosis. Herein, we report on the proof-of-concept of a simple method enabling in vitro detection of a structural spike protein subunit from the SARS-CoV-2 (S1 ) in aqueous samples. At the core of this discovery lies the well-known paradigm of monitoring the capacitive current across a reconstituted zwitterionic lipid membrane subjected to a periodic transmembrane potential, followed by the real-time spectral analysis enabling the extraction of the second harmonic of the capacitive current. Subsequent changes in the amplitude of this harmonic recorded during lipid membrane-S1 interactions were correlated with alterations induced in the inner membrane potential profile by the S1 protein subunit adsorption, and were shown to be augmented by ionic strength, the presence of a specific monoclonal antibody designed against the S1 subunit and the angiotensin-converting enzyme 2 (ACE2) protein receptor, and uninhibited by the presence of other human serum proteins.

The Nanopore-Tweezing-Based, Targeted Detection of Nucleobases on Short Functionalized Peptide Nucleic Acid Sequences.

The implication of nanopores as versatile components in dedicated biosensors, nanoreactors, or miniaturized sequencers has considerably advanced single-molecule investigative science in a wide range of disciplines, ranging from molecular medicine and nanoscale chemistry to biophysics and ecology. Here, we employed the nanopore tweezing technique to capture amino acid-functionalized peptide nucleic acids (PNAs) with α-hemolysin-based nanopores and correlated the ensuing stochastic fluctuations of the ionic current through the nanopore with the composition and order of bases in the PNAs primary structure. We demonstrated that while the system enables the detection of distinct bases on homopolymeric PNA or triplet bases on heteropolymeric strands, it also reveals rich insights into the conformational dynamics of the entrapped PNA within the nanopore, relevant for perfecting the recognition capability of single-molecule sequencing.

Non-Receptor-Mediated Lipid Membrane Permeabilization by the SARS-CoV-2 Spike Protein S1 Subunit.

Due to the pressing need to generate specific drugs or vaccines for COVID-19 and management of its outbreak, detailed knowledge regarding the SARS-CoV-2 entry into host cells and timely, cheap, and easy-to-use detection methods are of critical importance for containing the SARS-CoV-2 epidemic. Through electrophysiology and fluorescence spectroscopy experiments, we show that even in the absence of the angiotensin-converting enzyme 2 receptor, the S1 subunit from SARS-CoV-2 spike protein binding to neutral phospholipid membranes leads to their mechanical destabilization and permeabilization. A similar cytotoxic effect of the protein was seen in human lung epithelial cells. A monoclonal antibody generated toward the S1 subunit alleviates to a considerable extent the destabilizing potential of the protein in such model membranes. Finally, we demonstrate the proof-of-concept capability of an α-hemolysin (α-HL) protein nanopore to detect in aqueous buffer and real time the region-binding domain of the S1 subunit from SARS-CoV-2 spike protein by monitoring its immunological interaction with a target antibody. Our results may offer new perspectives in understanding the pathogenesis of the SARS-CoV-2 infection, its treatment, and real-time detection.

Author Correction: Sequence-specific detection of single-stranded DNA with a gold nanoparticle-protein nanopore approach.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Sequence-specific detection of single-stranded DNA with a gold nanoparticle-protein nanopore approach.

Fast, cheap and easy to use nucleic acids detection methods are crucial to mitigate adverse impacts caused by various pathogens, and are essential in forensic investigations, food safety monitoring or evolution of infectious diseases. We report here a method based on the α-hemolysin (α-HL) nanopore, working in conjunction to unmodified citrate anion-coated gold nanoparticles (AuNPs), to detect nanomolar concentrations of short single-stranded DNA sequences (ssDNA). The core idea was to use charge neutral peptide nucleic acids (PNA) as hybridization probe for complementary target ssDNAs, and monitor at the single-particle level the PNA-induced aggregation propensity AuNPs during PNA-DNA duplexes formation, by recording ionic current blockades signature of AuNP-α-HL interactions. This approach offers advantages including: (1) a simple to operate platform, producing clear-cut readout signals based on distinct size differences of PNA-induced AuNPs aggregates, in relation to the presence in solution of complementary ssDNAs to the PNA fragments (2) sensitive and selective detection of target ssDNAs (3) specific ssDNA detection in the presence of interference DNA, without sample labeling or signal amplification. The powerful synergy of protein nanopore-based nanoparticle detection and specific PNA-DNA hybridization introduces a new strategy for nucleic acids biosensing with short detection time and label-free operation.

Bee venom-derived antimicrobial peptide melectin has broad-spectrum potency, cell selectivity, and salt-resistant properties.

Antimicrobial peptides have attracted attention as alternatives to conventional antibiotics. Previously, a novel antimicrobial peptide, melectin, consisting of 18 amino acids was isolated from the venom of a bee, Melecta albifrons. Here, we investigated the antibacterial activity of melectin against drug-resistant bacteria. Melectin showed broad-spectrum antimicrobial activity but low cytotoxicity and no hemolytic activity. Melectin maintained its antimicrobial activity at physiological salt concentrations. Melectin is an α-helical structure that binds to the bacterial membrane via electrostatic interactions and kills bacteria in a short time by bacterial membrane targeting. Collectively, our results suggest that melectin has antibacterial activity and anti-inflammatory activity.

Unzipping Mechanism of Free and Polyarginine-Conjugated DNA-PNA Duplexes, Preconfined Inside the α-Hemolysin Nanopore.

In this work, comparative studies on DNA-PNA and polyarginine-conjugated DNA-PNA duplexes unzipping inside the α-hemolysin nanopore (α-HL) are presented. We identified significant differences in the blockade currents, as the applied voltage across the nanopore facilitated the duplex capture inside the nanopore's vestibule against the constriction region, subsequent cDNA strand insertion inside the nanopore's ß-barrel past the constriction site, its complete unzip from the duplex, and translocation. We observed that inside the voltage-biased nanopore, polyarginine-conjugated DNA-PNA duplexes dehybridize faster than their DNA-PNA counterparts and proposed a model to describe the duplex unzipping. This study identifies key particularities of DNA-PNA duplex unzipping as it takes place inside the nanopore and being preceded by entrapment in the vestibule domain of the α-HL. Our results are a crucial step toward understanding the nucleic acids duplexes unzipping kinetics variability, in confined, variable geometries.

Antimicrobial peptide HPA3NT3-A2 effectively inhibits biofilm formation in mice infected with drug-resistant bacteria.

Bacterial biofilms formed through secretion of extracellular polymeric substances (EPS) have been implicated in many serious infections and can increase antibiotic resistance by a factor of more than 1000. Here, we examined the abilities of the antimicrobial peptide HPA3NT3-A2 to inhibit and reduce biofilm formation, eliminate EPS, and suppress inflammation in mice infected with clinical isolates of drug-resistant Pseudomonas aeruginosa strains. HPA3NT3-A2 was developed from a desirable analogue peptide, HPA3NT3, derived from residues 2-20 of the Helicobacter pylori ribosomal protein L1. HPA3NT3-A2 showed stronger activity against planktonic cells (MIC: 8 µM) compared to ciprofloxacin or tobramycin (>512 µM), and a favorable minimum biofilm inhibition and elimination concentration. This peptide also neutralized LPS; decreased levels of EPS; inhibited the production of pro-inflammatory cytokines in the lung, kidney, and spleen; decreased white blood cell counts; and increased survival among infected mice.

Mechanism of action of antimicrobial peptide P5 truncations against Pseudomonas aeruginosa and Staphylococcus aureus.

Rates of microbial drug resistance are increasing worldwide; therefore, antimicrobial peptides (AMPs) are considered promising alternative therapeutic agents to antibiotics. AMPs are essential components of the innate immune system and exhibit broad-spectrum antimicrobial activity. P5 is a Cecropin A-Magainin 2 hybrid analog peptide with antimicrobial activity against Gram-negative and Gram-positive bacteria. In the present study, truncated peptides were designed to reduction length, retainment their antimicrobial activity and low toxicity at high concentrations compared with that of the parent peptide P5. The truncated peptides P5-CT1 and P5-NT1 exhibited antibacterial activities against both Gram-negative and Gram-positive bacteria. In contrast, P5-CT2, P5-CT3, P5-NT2, and P5-NT3 showed higher antibacterial activities against gram-positive bacteria compared to Gram-negative bacteria at low concentration of peptides. The truncated peptides showed lower hemolytic activity and toxic effects against mammalian cells compared with those of the parent peptide P5. The levels of several truncated peptides were maintained in the presence of physiological concentrations of salts, indicating their high stability. The results of flow cytometry, propidium iodide uptake, n-phenyl-1-naphthylamine uptake, and 3,3'-dipropylthiadicarbocyanine iodide assays showed that these truncated peptides killed microbial cells by increasing membrane permeability, thereby causing membrane damage. The results suggested that truncated peptides of P5 have good potential for use as novel antimicrobial agents.

Nanopore-Assisted, Sequence-Specific Detection, and Single-Molecule Hybridization Analysis of Short, Single-Stranded DNAs.

We report here on the ability of the α-hemolysin (α-HL) nanopore to achieve label-free, selective, and real-time detection of 15 nt long ssDNA fragments in solution, by exploiting their hybridization with freely added, polycationic peptides-functionalized PNAs. At the core of our work lies the paradigm that when PNAs and ssDNA are mixed together, the bulk concentration of free PNA decreases, depending upon the (mis)match degree between complementary strands and their relative concentrations. We demonstrate that the ssDNA sensing principle and throughput of the method are determined by the rate at which nonhybridized, polycationic peptides-functionalized PNA molecules arrive at the α-HL's vestibule entrance and thread into the nanopore. We found that with the application of a 30-fold salt gradient across the nanopore, the method enhances single-molecule detection sensitivity in the nanomolar range of ssDNA concentrations. This study demonstrates that the transmembrane potential-dependent unzip of single PNA-DNA duplexes at the α-HL's ß-barrel entry permits discrimination between sequences that differ by one base pair.

Nonfunctionalized PNAs as Beacons for Nucleic Acid Detection in a Nanopore System.

In this work, single-channel current recordings were used to selectively detect individual ssDNA strands in the vestibule of the α-hemolysin (α-HL) protein nanopore. The sensing mechanism was based on the detection of the intrinsic topological change of target ssDNA molecules after the hybridization with complementary PNA fragments. The readily distinguishable current signatures of PNA-DNA duplexes reversible association with the α-HL's vestibule, in terms of blockade amplitudes and kinetic features, allows specific detection of nucleic acid hybridization.