The emerging landscape of single-molecule protein sequencing technologies.

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

Heavy Water Reduces the Electronic Conductance of Protein Wires via Deuteron Interactions with Aromatic Residues.

Proteins are versatile, self-assembling nanoelectronic components, but their hopping conductivity is expected to be influenced by solvent fluctuations. The role of the solvent was investigated by measuring the single molecule conductance of several proteins in both H2O and D2O. The conductance of a homologous series of protein wires decreases more rapidly with length in D2O, indicating a 6-fold decrease in carrier diffusion constant relative to the same protein in H2O. The effect was found to depend on the specific aromatic amino acid composition. A tryptophan zipper protein showed a decrease in conductance similar to that of the protein wires, whereas a phenylalanine zipper protein was insensitive to solvent changes. Tryptophan contains an indole amine, whereas the phenylalanine aromatic ring has no exchangeable protons, so the effect of heavy water on conductance is a consequence of specific D- or H-interactions with the aromatic residues.

Systematics and biogeography of the Old World fern genus Antrophyum.

Antrophyum is one of the largest genera of vittarioid ferns (Pteridaceae) and is most diverse in tropical Asia and the Pacific Islands, but also occurs in temperate Asia, Australia, tropical Africa and the Malagasy region. The only monographic study of Antrophyum was published more than a century ago and a modern assessment of its diversity is lacking. Here, we reconstructed a comprehensively sampled and robustly supported phylogeny for the genus based on four chloroplast markers using Bayesian inference, maximum likelihood and maximum parsimony analyses. We then explored the evolution of the genus from the perspectives of morphology, systematics and historical biogeography. We investigated nine critical morphological characters using a morphometric approach and reconstructed their evolution on the phylogeny. We describe four new species and provide new insight into species delimitation. We currently recognize 34 species for the genus and provide a key to identify them. The results of biogeographical analysis suggest that the distribution of extant species is largely shaped by both ancient and recent dispersal events.

Role of contacts in long-range protein conductance.

Proteins are widely regarded as insulators, despite reports of electrical conductivity. Here we use measurements of single proteins between electrodes, in their natural aqueous environment to show that the factor controlling measured conductance is the nature of the electrical contact to the protein, and that specific ligands make highly selective electrical contacts. Using six proteins that lack known electrochemical activity, and measuring in a potential region where no ion current flows, we find characteristic peaks in the distributions of measured single-molecule conductances. These peaks depend on the contact chemistry, and hence, on the current path through the protein. In consequence, the measured conductance distribution is sensitive to changes in this path caused by ligand binding, as shown with streptavidin-biotin complexes. Measured conductances are on the order of nanosiemens over distances of many nanometers, orders of magnitude more than could be accounted for by electron tunneling. The current is dominated by contact resistance, so the conductance for a given path is independent of the distance between electrodes, as long as the contact points on the protein can span the gap between electrodes. While there is no currently known biological role for high electronic conductance, its dependence on specific contacts has important technological implications, because no current is observed at all without at least one strongly bonded contact, so direct electrical detection is a highly selective and label-free single-molecule detection method. We demonstrate single-molecule, highly specific, label- and background free-electronic detection of IgG antibodies to HIV and Ebola viruses.

Probing Bioelectronic Connections Using Streptavidin Molecules with Modified Valency.

As molecular electronic components, proteins are distinguished by a remarkably long electronic decay length (∼10 nm) together with high contact resistance and extreme sensitivity to the chemical details of the contact. As a consequence, the conductance of even a large bioelectronic assembly is largely controlled by the conductance of the contacts. Streptavidin is a versatile linker protein that can tether together biotinylated electrodes and biotinylated proteins but with an ambiguity about the contact geometry that arises from its four possible binding sites for biotin. Here, we use engineered streptavidin tetramers, selected to contain a defined ratio of active monomers to "dead" monomers so as to define the biotin binding sites. We find a strong dependence of conductance on the separation of the biotin molecules, consistent with a short-range tunneling interaction within the streptavidin and in contrast to the long-range transport observed inside larger proteins. Hexaglutamate tails label the active monomers, and the additional negative charge enhances conductance significantly. This effect is quantitatively accounted for by an electronic resonance in the protein conductance.

SIR proteins create compact heterochromatin fibers.

Heterochromatin is a silenced chromatin region essential for maintaining genomic stability and driving developmental processes. The complicated structure and dynamics of heterochromatin have rendered it difficult to characterize. In budding yeast, heterochromatin assembly requires the SIR proteins-Sir3, believed to be the primary structural component of SIR heterochromatin, and the Sir2-4 complex, responsible for the targeted recruitment of SIR proteins and the deacetylation of lysine 16 of histone H4. Previously, we found that Sir3 binds but does not compact nucleosomal arrays. Here we reconstitute chromatin fibers with the complete complement of SIR proteins and use sedimentation velocity, molecular modeling, and atomic force microscopy to characterize the stoichiometry and conformation of SIR chromatin fibers. In contrast to fibers with Sir3 alone, our results demonstrate that SIR arrays are highly compact. Strikingly, the condensed structure of SIR heterochromatin fibers requires both the integrity of H4K16 and an interaction between Sir3 and Sir4. We propose a model in which a dimer of Sir3 bridges and stabilizes two adjacent nucleosomes, while a Sir2-4 heterotetramer interacts with Sir3 associated with a nucleosomal trimer, driving fiber compaction.

Electronic Conductance Resonance in Non-Redox-Active Proteins.

Bioelectronics research has mainly focused on redox-active proteins because of their role in biological charge transport. In these proteins, electronic conductance is a maximum when electrons are injected at the known redox potential of the protein. It has been shown recently that many non-redox-active proteins are good electronic conductors, though the mechanism of conduction is not yet understood. Here, we report single-molecule measurements of the conductance of three non-redox-active proteins, maintained under potential control in solution, as a function of electron injection energy. All three proteins show a conductance resonance at a potential ∼0.7 V removed from the nearest oxidation potential of their constituent amino acids. If this shift reflects a reduction of reorganization energy in the interior of the protein, it would account for the long-range conductance observed when carriers are injected into the interior of a protein.

Electronic Decay Length in a Protein Molecule.

Antibodies have two identical binding domains and can therefore form a well-defined conducting bridge by binding a pair of electrodes functionalized with an epitope. The conductance measured between these two fixed points on the antibody does not change with the size of the electrode gap. A second conduction path is via one specific attachment to an epitope and a second nonspecific attachment to the surface of the antibody. In this case, the conductance does change with gap size, yielding an estimated electronic decay length >6 nm, long enough that it is not possible to distinguish between an exponential or a hyperbolic distance dependence. This decay length is substantially greater than that measured for hopping transport in an organic molecular wire.

A Y-Shaped Three-Arm Structure for Probing Bivalent Interactions between Protein Receptor-Ligand Using AFM and SPR.

The goal of this research was to develop linkage chemistry for the study of bivalent interactions between a receptor and its ligand using atomic force microscopy (AFM) and surface plasmon resonance (SPR). We conceived a three-arm structure composed of flexible chains connected to a large rigid core with orthogonal functional groups at their ends for formation and attachment (or immobilization) of bivalent ligands. To demonstrate the principle, we chose the well-known biotin-streptavidin interaction as a model system. On the basis of a crystal structure of the biotin-streptavidin complex, we designed and synthesized a bisbiotin ligand to have a Y shape with two biotin motifs on its arms for binding and a functional group on its stem for immobilization or attachment, referred to as y-bisbiotin. First, we found that the y-bisbiotin ligand stabilized the streptavidin more than its monobiotin counterpart did in solution, which indicates that the bivalent interaction was synergistic. The y-bisbiotin was attached to AFM tips through a click reaction for the force measurement experiments, which showed that unbinding the bisbiotin from streptavidin needed twice the force of unbinding a monobiotin. For the SPR study, we added a ω-thiolated alkyl chain to y-bisbiotin for its incorporation into a monolayer. The SPR data indicated that the streptavidin dissociated from a mixed monolayer bearing y-bisbiotin much slower than from the one bearing monobiotin. This work demonstrates unique chemistry for the study of bivalent interactions using AFM and SPR.

Recent Progress in Molecular Recognition Imaging Using Atomic Force Microscopy.

Atomic force microscopy (AFM) is an extremely powerful tool in the field of bionanotechnology because of its ability to image single molecules and make measurements of molecular interaction forces with piconewton sensitivity. It works in aqueous media, enabling studies of molecular phenomenon taking place under physiological conditions. Samples can be imaged in their near-native state without any further modifications such as staining or tagging. The combination of AFM imaging with the force measurement added a new feature to the AFM technique, that is, molecular recognition imaging. Molecular recognition imaging enables mapping of specific interactions between two molecules (one attached to the AFM tip and the other to the imaging substrate) by generating simultaneous topography and recognition images (TREC). Since its discovery, the recognition imaging technique has been successfully applied to different systems such as antibody-protein, aptamer-protein, peptide-protein, chromatin, antigen-antibody, cells, and so forth. Because the technique is based on specific binding between the ligand and receptor, it has the ability to detect a particular protein in a mixture of proteins or monitor a biological phenomenon in the native physiological state. One key step for recognition imaging technique is the functionalization of the AFM tips (generally, silicon, silicon nitrides, gold, etc.). Several different functionalization methods have been reported in the literature depending on the molecules of interest and the material of the tip. Polyethylene glycol is routinely used to provide flexibility needed for proper binding as a part of the linker that carries the affinity molecule. Recently, a heterofunctional triarm linker has been synthesized and successfully attached with two different affinity molecules. This novel linker, when attached to AFM tip, helped to detect two different proteins simultaneously from a mixture of proteins using a so-called "two-color" recognition image. Biological phenomena in nature often involve multimolecular interactions, and this new linker could be ideal for studying them using AFM recognition imaging. It also has the potential to be used extensively in the diagnostics technique. This Account includes fundamentals behind AFM recognition imaging, a brief discussion on tip functionalization, recent advancements, and future directions and possibilities.

Electrophoretic Deformation of Individual Transfer RNA Molecules Reveals Their Identity.

It has been hypothesized that the ribosome gains additional fidelity during protein translation by probing structural differences in tRNA species. We measure the translocation kinetics of different tRNA species through ∼3 nm diameter synthetic nanopores. Each tRNA species varies in the time scale with which it is deformed from equilibrium, as in the translocation step of protein translation. Using machine-learning algorithms, we can differentiate among five tRNA species, analyze the ratios of tRNA binary mixtures, and distinguish tRNA isoacceptors.

A three-arm scaffold carrying affinity molecules for multiplex recognition imaging by atomic force microscopy: the synthesis, attachment to silicon tips, and detection of proteins.

We have developed a multiplex imaging method for detection of proteins using atomic force microscopy (AFM), which we call multiplex recognition imaging (mRI). AFM has been harnessed to identify protein using a tip functionalized with an affinity molecule at a single molecule level. However, many events in biochemistry require identification of colocated factors simultaneously, and this is not possible with only one type of affinity molecule on an AFM tip. To enable AFM detection of multiple analytes, we designed a recognition head made from conjugating two different affinity molecules to a three-arm linker. When it is attached to an AFM tip, the recognition head would allow the affinity molecules to function in concert. In the present study, we synthesized two recognition heads: one was composed of two nucleic acid aptamers, and the other one composed of an aptamer and a cyclic peptide. They were attached to AFM tips through a catalyst-free click reaction. Our imaging results show that each affinity unit in the recognition head can recognize its respective cognate in an AFM scanning process independently and specifically. The AFM method was sensitive, only requiring 2 to 3 µL of protein solution with a concentration of ∼2 ng/mL for the detection with our current setup. When a mixed sample was deposited on a surface, the ratio of proteins could be determined by counting numbers of the analytes. Thus, this mRI approach has the potential to be used as a label-free system for detection of low-abundance protein biomarkers.

Physical model for recognition tunneling.

Recognition tunneling (RT) identifies target molecules trapped between tunneling electrodes functionalized with recognition molecules that serve as specific chemical linkages between the metal electrodes and the trapped target molecule. Possible applications include single molecule DNA and protein sequencing. This paper addresses several fundamental aspects of RT by multiscale theory, applying both all-atom and coarse-grained DNA models: (1) we show that the magnitude of the observed currents are consistent with the results of non-equilibrium Green's function calculations carried out on a solvated all-atom model. (2) Brownian fluctuations in hydrogen bond-lengths lead to current spikes that are similar to what is observed experimentally. (3) The frequency characteristics of these fluctuations can be used to identify the trapped molecules with a machine-learning algorithm, giving a theoretical underpinning to this new method of identifying single molecule signals.

Application of catalyst-free click reactions in attaching affinity molecules to tips of atomic force microscopy for detection of protein biomarkers.

Atomic force microscopy (AFM) has been extensively used in studies of biological interactions. Particularly, AFM based force spectroscopy and recognition imaging can sense biomolecules on a single molecule level, having great potential to become a tool for molecular diagnostics in clinics. These techniques, however, require affinity molecules to be attached to AFM tips in order to specifically detect their targets. The attachment chemistry currently used on silicon tips involves multiple steps of reactions and moisture sensitive chemicals, such as (3-aminopropyl)triethoxysilane (APTES) and N-hydroxysuccinimide (NHS) ester, making the process difficult to operate in aqueous solutions. In the present study, we have developed a user-friendly protocol to functionalize the AFM tips with affinity molecules. A key feature of it is that all reactions are carried out in aqueous solutions. In summary, we first synthesized a molecular anchor composed of cyclooctyne and silatrane for introduction of a chemically reactive function to AFM tips and a bifunctional polyethylene glycol linker that harnesses two orthogonal click reactions, copper free alkyne-azide cycloaddition and thiol-vinylsulfone Michael addition, for attaching affinity molecules to AFM tips. The attachment chemistry was then validated by attaching antithrombin DNA aptamers and cyclo-RGD peptides to silicon nitride (SiN) tips, respectively, and measuring forces of unbinding these affinity molecules from their protein cognates human α-thrombin and human α5ß1-integrin immobilized on mica surfaces. In turn, we used the same attachment chemistry to functionalize silicon tips with the same affinity molecules for AFM based recognition imaging, showing that the disease-relevant biomarkers such as α-thrombin and α5ß1-integrin can be detected with high sensitivity and specificity by the single molecule technique. These studies demonstrate the feasibility of our attachment chemistry for the use in functionalization of AFM tips with affinity molecules.

Long-Range Conductivity in Proteins Mediated by Aromatic Residues.

Single-molecule measurements show that many proteins, lacking any redox cofactors, nonetheless exhibit electrical conductance on the order of a nanosiemen over 10 nm distances, implying that electrons can transit an entire protein in less than a nanosecond when subject to a potential difference of less than 1 V. This is puzzling because, for fast transport (i.e., a free energy barrier of zero), the hopping rate is determined by the reorganization energy of approximately 0.8 eV, and this sets the time scale of a single hop to at least 1 µs. Furthermore, the Fermi energies of typical metal electrodes are far removed from the energies required for sequential oxidation and reduction of the aromatic residues of the protein, which should further reduce the hopping current. Here, we combine all-atom molecular dynamics (MD) simulations of non-redox-active proteins (consensus tetratricopeptide repeats) with an electron transfer theory to demonstrate a molecular mechanism that can account for the unexpectedly fast electron transport. According to our MD simulations, the reorganization energy produced by the energy shift on charging (the Stokes shift) is close to the conventional value of 0.8 eV. However, the non-ergodic sampling of molecular configurations by the protein results in reaction-reorganization energies, extracted directly from the distribution of the electrostatic energy fluctuations, that are only ∼0.2 eV, which is small enough to enable long-range conductivity, without invoking quantum coherent transport. Using the MD values of the reorganization energies, we calculate a current decay with distance that is in agreement with experiment.

Long lifetime of hydrogen-bonded DNA basepairs by force spectroscopy.

Electron-tunneling data suggest that a noncovalently-bonded complex of three molecules, two recognition molecules that present hydrogen-bond donor and acceptor sites via a carboxamide group, and a DNA base, remains bound for seconds. This is surprising, given that imino-proton exchange rates show that basepairs in a DNA double helix open on millisecond timescales. The long lifetime of the three-molecule complex was confirmed using force spectroscopy, but measurements on DNA basepairs are required to establish a comparison with the proton-exchange data. Here, we report on a dynamic force spectroscopy study of complexes between the bases adenine and thymine (A-T, two-hydrogen bonds) and 2-aminoadenine and thymine (2AA-T, three-hydrogen bonds). Bases were tethered to an AFM probe and mica substrate via long, covalently linked polymer tethers. Data for bond-survival probability versus force and the rupture-force distributions were well fitted by the Bell model. The resulting lifetime of the complexes at zero pulling force was ~2 s for two-hydrogen bonds (A-T) and ~4 s for three-hydrogen bonds (2AA-T). Thus, DNA basepairs in an AFM pulling experiment remain bonded for long times, even without the stabilizing influence of base-stacking in a double helix. This result suggests that the pathways for opening, and perhaps the open states themselves, are very different in the AFM and proton-exchange measurements.

Hydrophobicity of methylated DNA as a possible mechanism for gene silencing.

AFM images show that chromatin reconstituted on methylated DNA (meDNA) is compacted when imaged under water. Chromatin reconstituted on unmethylated DNA is less compacted and less sensitive to hydration. These differences must reflect changes in the physical properties of DNA on methylation, but prior studies have not revealed large differences between methylated and unmethylated DNA. Quasi-elastic light scattering studies of solutions of methylated and unmethylated DNA support this view. In contrast, AFM images of molecules at a water/solid interface yield a persistence length that nearly doubles (to 92.5 ± 4 nm) when 9% of the total DNA is methylated. This increase in persistence length is accompanied by a decrease in contour length, suggesting that a significant fraction of the meDNA changes into the stiffer A form as the more hydrophobic meDNA is dehydrated at the interface. This suggests a simple mechanism for gene silencing as the stiffer meDNA is more difficult to remove from nucleosomes.

Synthesis, physicochemical properties, and hydrogen bonding of 4(5)-substituted 1-H-imidazole-2-carboxamide, a potential universal reader for DNA sequencing by recognition tunneling.

We have developed a chemical reagent that recognizes all naturally occurring DNA bases, a so called universal reader, for DNA sequencing by recognition tunneling in nanopores.1 The primary requirements for this type of molecules are the ability to form non-covalent complexes with individual DNA bases and to generate recognizable electronic signatures under an electrical bias. 1-H-imidazole-2-carboxamide was designed as such a recognition moiety to interact with the DNA bases through hydrogen bonding. In the present study, we first furnished a synthetic route to 1-H-imidazole-2-carboxamide containing a short ω-functionalized alkyl chain at its 4(5) position for its attachment to metal and carbon electrodes. The acid dissociation constants of the imidazole-2-carboxamide were then determined by UV spectroscopy. The data show that the 1-H-imidazole-2-carboxamide exists in a neutral form between pH 6-10. Density functional theory (DFT) and NMR studies indicate that the imidazole ring exists in prototropic tautomers. We propose an intramolecular mechanism for tautomerization of 1-H-imidazole-2-carboxamide. In addition, the imidazole-2-carboxamide can self-associate to form hydrogen bonded dimers. NMR titration found that naturally occurring nucleosides interacted with 1-H-imidazole-2-carboxamide through hydrogen bonding in a tendency of dG>dC≫dT>dA. These studies are indispensable to assisting us in understanding the molecular recognition that takes place in the nanopore where routinely used analytical tools such as NMR and FTIR cannot be conveniently applied.

1,8-Naphthyridine-2,7-diamine: a potential universal reader of Watson-Crick base pairs for DNA sequencing by electron tunneling.

With the aid of Density Functional Theory (DFT), we designed 1,8-naphthyridine-2,7-diamine as a recognition molecule to read DNA base pairs for genomic sequencing by electron tunneling. NMR studies show that it can form stable triplets with both A : T and G : C base pairs through hydrogen bonding. Our results suggest that the naphthyridine molecule should be able to function as a universal base pair reader in a tunneling gap, generating distinguishable signatures under electrical bias for each of DNA base pairs.

DNA translocating through a carbon nanotube can increase ionic current.

Translocation of DNA through a narrow, single-walled carbon nanotube can be accompanied by large increases in ion current, recently observed in contrast to the ion current blockade. We use molecular dynamics simulations to show that large electro-osmotic flow can be turned into a large net current via ion-selective filtering by a DNA molecule inside the carbon nanotube.