Analyte‐Triggered DNA‐Probe Release from a Triplex Molecular Beacon for Nanopore Sensing

A new nanopore sensing strategy based on triplex molecular beacon was developed for the detection of specific DNA or multivalent proteins. The sensor is composed of a triplex‐forming molecular beacon and a stem‐forming DNA component that is modified with a host–guest complex. Upon target DNA hybridizing with the molecular beacon loop or multivalent proteins binding to the recognition elements on the stem, the DNA probe is released and produces highly characteristic current signals when translocated through α‐hemolysin. The frequency of current signatures can be used to quantify the concentrations of the target molecules. This sensing approach provides a simple, quick, and modular tool for the detection of specific macromolecules with high sensitivity and excellent selectivity. It may find useful applications in point‐of‐care diagnostics with a portable nanopore kit in the future.     

Protein Detection Through Single Molecule Nanopore

A single nanopore represents a versatile single-molecule probe that can be employed to reveal several important features of proteins, such as physical structure, backbone flexibility, mechanical stability, their folding state, binding affinity to other interacting ligands and enzymatic activity. In this review, we summarize the development and current research related to the field of protein detection by nanopore, as well as a few examples of the pioneer work on protein detection. We first discuss the principle of electrical detection with nanopores and how this technique provides information from current traces. Then the development from peptide detection with biological nanopore to protein detection through solid-state nanopore is described. Finally, we prospect the measurement of protein shape and construction using nanopore technology for the applications in life research area.     

Discrimination of single‐stranded DNA homopolymers by sieving out G‐quadruplex using tiny solid‐state nanopores

Nanopore sensor has been developed as a promising technology for DNA sequencing at the single‐base resolution. However, the discrimination of homopolymers composed of guanines from other nucleotides has not been clearly revealed due to the easily formed G‐quadruplex in aqueous buffers. In this work, we report that a tiny silicon nitride nanopore was used to sieve out G tetramers to make sure only homopolymers composed of guanines could translocate through the nanopore, then the 20‐nucleotide long ssDNA homopolymers could be identified and differentiated. It is found that the size of the nucleotide plays a major role in affecting the current blockade as well as the dwell time while DNA is translocating through the nanopore. By the comparison of translocation behavior of ssDNA homopolymers composed of nucleotides with different volumes, it is found that smaller nucleotides can lead to higher translocation speed and lower current blockage, which is also found and validated for the 105‐nucleotide long homopolymers. The studies performed in this work will improve our understanding of nanopore‐based DNA sequencing at single‐base level.     

Recognition-domain focused chemosensors: versatile and efficient reporters of protein kinase activity

Catalyzed by kinases, serine/threonine and tyrosine phosphorylation is a vital mechanism of intracellular regulation. Thus, assays that easily monitor kinase activity are critical in both academic and pharmaceutical settings. We previously developed sulfonamido-oxine (Sox)-based fluorescent peptides following a beta-turn focused (BTF) design for the continuous assay of kinase activity in vitro and in cell lysates. Upon phosphorylation of the Sox-containing peptide, the chromophore binds Mg (2+) and undergoes chelation-enhanced fluorescence (CHEF). Although the design was applied successfully to the development of several kinase sensors, an intrinsic limitation was that only residues C- or N-terminal to the phosphorylated residue could be used to derive specificity for the target kinase. To address this limitation, a new, recognition-domain focused (RDF) strategy was developed that also relies on CHEF. In this approach, the requirement for the constrained beta-turn motif is obviated by alkylation of a cysteine residue with a Sox-based derivative to afford an amino acid termed C-Sox. The RDF design allows inclusion of extended binding determinants to maximize recognition by the cognate kinase, which has now permitted the construction of chemosensors for a variety of representative Ser/Thr (PKC alpha, PKC betaIota, PKC delta, Pim2, Akt1, MK2, and PKA) as well as receptor (IRK) and nonreceptor (Src, Abl) Tyr kinases with greatly enhanced selectivity. The new sensors have up to 28-fold improved catalytic efficiency and up to 66-fold lower K M when compared to the corresponding BTF probes. The improved generality of the strategy is exemplified with the synthesis and analysis of Sox-based probes for PKC betaIota and PKC delta, which were previously unattainable using the BTF approach.     

Reversing current rectification to improve DNA‐sensing sensitivity in conical nanopores

Herein, we report the ultrasensitive DNA detection through designing an elegant nanopore biosensor as the first case to realize the reversal of current rectification direction for sensing. Attributed to the unique asymmetric structure, the glass conical nanopore exhibits the sensitive response to the surface charge, which can be facilely monitored by ion current rectification curves. In our design, an enzymatic cleavage reaction was employed to alter the surface charge of the nanopore for DNA sensing. The measured ion current rectification was strongly responsive to DNA concentrations, even reaching to the reversed status from the negative ratio (?6.5) to the positive ratio (+16.1). The detectable concentration for DNA was as low as 0.1 fM. This is an ultrasensitive and label‐free DNA sensing approach, based on the rectification direction‐reversed amplification in a single glass conical nanopore.     

Voltage-controlled metal binding on polyelectrolyte-functionalized nanopores

Most of the research in the field of nanopore-based platforms is focused on monitoring ion currents and forces as individual molecules translocate through the nanopore. Molecular gating, however, can occur when target analytes interact with receptors appended to the nanopore surface. Here we show that a solid state nanopore functionalized with polyelectrolytes can reversibly bind metal ions, resulting in a reversible, real-time signal that is concentration dependent. Functionalization of the sensor is based on electrostatic interactions, requires no covalent bond formation, and can be monitored in real time. Furthermore, we demonstrate how the applied voltage can be employed to tune the binding properties of the sensor. The sensor has wide-ranging applications and, its simplest incarnation can be used to study binding thermodynamics using purely electrical measurements with no need for labeling.     

Protein detection by nanopores equipped with aptamers

Protein nanopores have been used as stochastic sensors for the detection of analytes that range from small molecules to proteins. In this approach, individual analyte molecules modulate the ionic current flowing through a single nanopore. Here, a new type of stochastic sensor based on an αHL pore modified with an aptamer is described. The aptamer is bound to the pore by hybridization to an oligonucleotide that is attached covalently through a disulfide bond to a single cysteine residue near a mouth of the pore. We show that the binding of thrombin to a 15-mer DNA aptamer, which forms a cation-stabilized quadruplex, alters the ionic current through the pore. The approach allows the quantification of nanomolar concentrations of thrombin, and provides association and dissociation rate constants and equilibrium dissociation constants for thrombin·aptamer interactions. Aptamer-based nanopores have the potential to be integrated into arrays for the parallel detection of multiple analytes.     

DNA‐Based Nanopore Sensing

Nanopore sensing is an attractive, label‐free approach that can measure single molecules. Although initially proposed for rapid and low‐cost DNA sequencing, nanopore sensors have been successfully employed in the detection of a wide variety of substrates. Early successes were mostly achieved based on two main strategies by 1) creating sensing elements inside the nanopore through protein mutation and chemical modification or 2) using molecular adapters to enhance analyte recognition. Over the past five years, DNA molecules started to be used as probes for sensing rather than substrates for sequencing. In this Minireview, we highlight the recent research efforts of nanopore sensing based on DNA‐mediated characteristic current events. As nanopore sensing is becoming increasingly important in biochemical and biophysical studies, DNA‐based sensing may find wider applications in investigating DNA‐involving biological processes.     

Mutant tyrosine kinases with unnatural nucleotide specificity retain the structure and phospho-acceptor specificity of the wild-type enzyme

The direct substrates of one protein kinase in a cell can be identified by mutation of the ATP binding pocket to allow an unnatural ATP analog to be accepted exclusively by the engineered kinase. Here, we present structural and functional assessment of peptide specificity of mutant protein kinases with unnatural ATP analogs. The crystal structure (2.8 A resolution) of c-Src (T338G) with N(6)-(benzyl) ADP bound shows that the creation of a unique nucleotide binding pocket does not alter the phospho-acceptor binding site of the kinase. A panel of optimal peptide substrates of defined sequence, as well as a degenerate peptide library, was utilized to assess the phospho-acceptor specificity of the engineered "traceable" kinases. The specificity profiles for the mutant kinases were found to be identical to those of their wild-type counterparts.     

Biosensing with conically shaped nanopores and nanotubes

In this review we consider recent results from our group that are directed towards developing "smart" synthetic nanopores that can mimic the functions of biological nanopores (transmembrane proteins). We first discuss the preparation and characterization of conical nanopores synthesized using the track-etch process. We then consider the design and function of conical nanopores that can rectify the ionic current that flows through these pores under an applied transmembrane potential. Finally, two types of sensors that we have developed with these conical nanopores are described. The first sensor makes use of molecular recognition elements that are bound to the nanopore mouth to selectively block the nanopore tip, thus detecting the presence of the analyte. The second sensor makes use of conical nanopores in a resistive-pulse type experiment, detecting the analyte via transient blockages in ionic current.     

Rapid access to unexplored chemical space by ligand scanning around a ruthenium center: discovery of potent and selective protein kinase inhibitors

An important objective for the discovery of compounds with unique biological activities is the development of methods for the synthesis of molecular scaffolds with defined three-dimensional shapes. We are currently investigating the scope of using metal complexes to accomplish this goal. In these compounds, the metal center has the role of organizing the orientation of the organic ligands, thus defining the overall shape of the molecule. A strategy is presented that allows a rapid scanning of ligands around a ruthenium center in the search for ligand spheres that are complementary in shape and functional group presentation to ATP binding sites of protein kinases. Following this approach, we have identified octahedral ruthenium complexes as potent inhibitors for the protein kinases Pim1, MSK1, and GSK3alpha.     

A Universal Strategy for Aptamer‐Based Nanopore Sensing through Host–Guest Interactions inside α‐Hemolysin

Nanopore emerged as a powerful single‐molecule technique over the past two decades, and has shown applications in the stochastic sensing and biophysical studies of individual molecules. Here, we report a versatile strategy for nanopore sensing by employing the combination of aptamers and host–guest interactions. An aptamer is first hybridized with a DNA probe which is modified with a ferrocene?cucurbit[7]uril complex. The presence of analytes causes the aptamer–probe duplex to unwind and release the DNA probe which can quantitatively produce signature current events when translocated through an α‐hemolysin nanopore. The integrated use of magnetic beads can further lower the detection limit by approximately two to three orders of magnitude. Because aptamers have shown robust binding affinities with a wide variety of target molecules, our proposed strategy should be universally applicable for sensing different types of analytes with nanopore sensors.     

Development of a peptide inhibitor-based cantilever sensor assay for cyclic adenosine monophosphate-dependent protein kinase

A highly sensitive nanomechanical cantilever sensor assay based on an electrical measurement has been developed for detecting activated cyclic adenosine monophosphate (cyclic AMP)-dependent protein kinase (PKA). Employing a peptide derived from the heat-stable protein kinase inhibitor (PKI), a magnetic bead system was first selected as a vehicle to immobilize the PKI-(5-24) peptide for capturing PKA catalytic subunit and the activity assay was applied for indirectly assessing the binding. Synergistic interactions of adenosine triphosphate (ATP) and the peptide inhibitor with the kinase were then investigated by a solution phase capillary electrophoretic assay, and by surface plasmon resonance technology which involved immobilization of the peptide inhibitor. After systemically evaluated by a homogeneous direct binding assay, the ATP-dependent recognition of the catalytic subunit of PKA by PKI-(5-24) was successfully transferred on to the nanomechanical cantilevers at protein concentrations of 6.6 pM-66 nM, exhibiting much higher sensitivity and wider dynamic range than the conventional activity assay. Thus, direct assessment of activated kinases using the cantilever sensor system functionalized with specific peptide inhibitors holds great promise in analytical applications and clinical medicine.     

Enzymatically modified peptide surfaces: towards general electrochemical sensor platform for protein kinase catalyzed phosphorylations

We hereby present an electrochemical approach for monitoring the three protein kinases sarcoma-related kinase (Src), extracellular signal-regulated kinase 1 (Erk1), and cyclin A-dependent kinase 2 (CDK2/cyclin A). The electrochemical sensor is based on the ability of kinases to transfer a redox-labeled phosphoryl group to surface-bound peptides that are highly specific substrates for the particular protein kinase (EGIYDVP, EPLTPSG, and HHASPRK, respectively). The detection method relies on the use of 5'-γ-ferrocenoyl-ATP (Fc-ATP) as a co-substrate for peptide phosphorylation. The peptides themselves are attached to a Au substrate, which acts as the working electrode. In this process a Fc-phosphoryl group is transferred to the peptide and the presence of the redox active Fc group is detected electrochemically. All peptide films were fully characterized by cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS). Particular attention was given to the electron transfer rates, k(ET), in peptide films after Fc-phosphorylation which were found to be on the order of seconds. The slow ET kinetics is presumably a result of the negative charge on the phosphoryl group. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) and X-ray photoelectron spectroscopy (XPS) experiments based on the peptide modified Au surfaces reveal significant ferrocene and phosphate group content introduced using the kinase-catalyzed phosphorylation reaction.     

Rapid determination of multiple linear kinase substrate motifs by mass spectrometry

Kinase-substrate recognition depends on the chemical properties of the phosphorylatable residue as well as the surrounding linear sequence motif. Detailed knowledge of these characteristics increases the confidence of linking identified phosphorylation sites to kinases, predicting phosphorylation sites, and designing optimal peptide substrates. Here, we present a mass spectrometry-based approach for determining linear kinase substrate motifs by elaborating the positional and chemical preference of the kinase for a phosphorylatable residue using libraries of naturally-occurring peptides that are amenable to peptide identification by commonly used proteomics platforms. We applied this approach to a structurally and functionally diverse set of purified kinases, which recapitulated their previously described substrate motifs and discovered additional ones, including preferences of certain kinases for phosphorylatable residues adjacent to peptide termini. Furthermore, we identify specific and distinguishable motif elements for the four members of the polo-like kinase (Plk) family and verify members of these motif elements for Plk1 in vivo.     

Single-molecule observation of the catalytic subunit of cAMP-dependent protein kinase binding to an inhibitor peptide

An engineered version of the staphylococcal alpha-hemolysin protein pore, bearing a peptide inhibitor near the entrance to the beta barrel, interacts with the catalytic (C) subunit of cAMP-dependent protein kinase. By monitoring the ionic current through the pore, binding events are detected at the single-molecule level. The kinetic and thermodynamic constants governing the binding interaction and the synergistic effect of MgATP are comparable but not identical to the values in bulk solution. Further, the values are strongly dependent on the applied membrane potential. Additional exploration of these findings may lead to a better understanding of the properties of enzymes at the lipid/water interface. Despite the complications, we suggest that the engineered pore might be used as a sensor element to screen inhibitors that act at either the substrate or ATP binding sites of the C subunit.     

Spatial blockage of ionic current for electrophoretic translocation of DNA through a graphene nanopore

Graphene nanopore has been promising the ultra‐high resolution for DNA sequencing due to the atomic thickness and excellent electronic properties of the graphene monolayer. The dynamical translocation phenomena and/or behaviors underneath the blocked ionic current, however, have not been well unveiled to date for the translocation of DNA electrophoretically through a graphene nanopore. In this report, the assessment on the sensitivity of ionic current to instantaneous statuses of DNA in a 2.4 nm graphene nanopore was carried out based on the all‐atom molecular dynamics simulations. By filtering out the thermal noise of ionic current, the instantaneous conformational variations of DNA in a graphene nanopore have been unveiled from the fluctuations of ionic current, because of the spatial blockage effect of DNA against ionic current. Interestingly, the neighborhood effect of DNA against ionic current was also observed within a distance of 1.5 nm nearby the graphene nanopore, suggesting the further precise control for DNA translocation through a graphene nanopore in gene sequencing. Moreover, the sensitivity of the blocked ionic current toward the instantaneous conformations of DNA in a graphene nanopore demonstrates the great potential of graphene nanopores in the dynamics analysis of single molecules.     

An Engineered OmpG Nanopore with Displayed Peptide Motifs for Single-Molecule Multiplex Protein Detection

Molecular detection via nanopore, achieved by monitoring changes in ionic current arising from analyte interaction with the sensor pore, is a promising technology for multiplex sensing development. Outer Membrane Protein G (OmpG), a monomeric porin possessing seven functionalizable loops, has been reported as an effective sensing platform for selective protein detection. Using flow cytometry to screen unfavorable constructs, we identified two OmpG nanopores with unique peptide motifs displayed in either loop 3 or 6, which also exhibited distinct analyte signals in single-channel current recordings. We exploited these motif-displaying loops concurrently to facilitate single-molecule multiplex protein detection in a mixture. We additionally report a strategy to increase sensor sensitivity via avidity motif display. These sensing schemes may be expanded to more sophisticated designs utilizing additional loops to increase multiplicity and sensitivity.     

Phosphorylation‐Responsive Membrane Transport of Peptides

Phosphorylation and dephosphorylation of peptides by kinases and phosphatases is essential for signal transduction in biological systems, and many diseases involve abnormal activities of these enzymes. Herein, we introduce amphiphilic calixarenes as key components for supramolecular, phosphorylation‐responsive membrane transport systems. Dye‐efflux experiments with liposomes demonstrated that calixarenes are highly active counterion activators for established cell‐penetrating peptides, with EC₅₀ values in the low nanomolar range. We have now found that they can even activate membrane transport of short peptide substrates for kinases involved in signal transduction, whereas the respective phosphorylated products are much less efficiently transported. This allows regulation of membrane transport activity by protein kinase A (PKA) and protein kinase C (PKC), as well as monitoring of their activity in a label‐free kinase assay.     

A coarse‐grained MARTINI‐like force field for DNA unzipping in nanopores

In nanopore force spectroscopy (NFS) a charged polymer is threaded through a channel of molecular dimensions. When an electric field is applied across the insulating membrane, the ionic current through the nanopore reports on polymer translocation, unzipping, dissociation, and so forth. We present a new model that can be applied in molecular dynamics simulations of NFS. Although simplified, it does reproduce experimental trends and all‐atom simulations. The scaled conductivities in bulk solution are consistent with experimental results for NaCl for a wide range of electrolyte concentrations and temperatures. The dependence of the ionic current through a nanopore on the applied voltage is symmetric and, in the voltage range used in experiments (up to 2 V), linear and in good agreement with experimental data. The thermal stability and geometry of DNA is well represented. The model was applied to simulations of DNA hairpin unzipping in nanopores. The results are in good agreement with all‐atom simulations: the scaled translocation times and unzipping sequence are similar. © 2015 Wiley Periodicals, Inc.