Electronic Properties of DNA Origami Nanostructures Revealed by In Silico Calculations.

DNA origami is a pioneering approach for producing complex 2- or 3-D shapes for use in molecular electronics due to its inherent self-assembly and programmability properties. The electronic properties of DNA origami structures are not yet fully understood, limiting the potential applications. Here, we conduct a theoretical study with a combination of molecular dynamics, first-principles, and charge transmission calculations. We use four separate single strand DNAs, each having 8 bases (4 × G4C4 and 4 × A4T4), to form two different DNA nanostructures, each having two helices bundled together with one crossover. We also generated double-stranded DNAs to compare electronic properties to decipher the effects of crossovers and bundle formations. We demonstrate that density of states and band gap of DNA origami depend on its sequence and structure. The crossover regions could reduce the conductance due to a lack of available states near the HOMO level. Furthermore, we reveal that, despite having the same sequence, the two helices in the DNA origami structure could exhibit different electronic properties, and electrode position can affect the resulting conductance values. Our study provides better understanding of the electronic properties of DNA origamis and enables us to tune these properties for electronic applications such as nanowires, switches, and logic gates.

Identifying SARS-CoV-2 Variants Using Single-Molecule Conductance Measurements.

The global COVID-19 pandemic has highlighted the need for rapid, reliable, and efficient detection of biological agents and the necessity of tracking changes in genetic material as new SARS-CoV-2 variants emerge. Here, we demonstrate that RNA-based, single-molecule conductance experiments can be used to identify specific variants of SARS-CoV-2. To this end, we (i) select target sequences of interest for specific variants, (ii) utilize single-molecule break junction measurements to obtain conductance histograms for each sequence and its potential mutations, and (iii) employ the XGBoost machine learning classifier to rapidly identify the presence of target molecules in solution with a limited number of conductance traces. This approach allows high-specificity and high-sensitivity detection of RNA target sequences less than 20 base pairs in length by utilizing a complementary DNA probe capable of binding to the specific target. We use this approach to directly detect SARS-CoV-2 variants of concerns B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron) and further demonstrate that the specific sequence conductance is sensitive to nucleotide mismatches, thus broadening the identification capabilities of the system. Thus, our experimental methodology detects specific SARS-CoV-2 variants, as well as recognizes the emergence of new variants as they arise.

Self-Aligning Nanojunctions for Integrated Single-Molecule Circuits.

Robust, high-yield integration of nanoscale components such as graphene nanoribbons, nanoparticles, or single-molecules with conventional electronic circuits has proven to be challenging. This difficulty arises because the contacts to these nanoscale devices must be precisely fabricated with angstrom-level resolution to make reliable connections, and at manufacturing scales this cannot be achieved with even the highest-resolution lithographic tools. Here we introduce an approach that circumvents this issue by precisely creating nanometer-scale gaps between metallic carbon electrodes by using a self-aligning, solution-phase process, which allows facile integration with conventional electronic systems with yields approaching 50%. The electrode separation is controlled by covalently binding metallic single-walled carbon nanotube (mCNT) electrodes to individual DNA duplexes to create mCNT-DNA-mCNT nanojunctions, where the gap is precisely matched to the DNA length. These junctions are then integrated with top-down lithographic techniques to create single-molecule circuits that have electronic properties dominated by the DNA in the junction, have reproducible conductance values with low dispersion, and are stable and robust enough to be utilized as active, high-specificity electronic biosensors for dynamic single-molecule detection of specific oligonucleotides, such as those related to the SARS-CoV-2 genome. This scalable approach for high-yield integration of nanometer-scale devices will enable opportunities for manufacturing of hybrid electronic systems for a wide range of applications.

DNA nanopores as artificial membrane channels for bioprotonics.

Biological membrane channels mediate information exchange between cells and facilitate molecular recognition. While tuning the shape and function of membrane channels for precision molecular sensing via de-novo routes is complex, an even more significant challenge is interfacing membrane channels with electronic devices for signal readout, which results in low efficiency of information transfer - one of the major barriers to the continued development of high-performance bioelectronic devices. To this end, we integrate membrane spanning DNA nanopores with bioprotonic contacts to create programmable, modular, and efficient artificial ion-channel interfaces. Here we show that cholesterol modified DNA nanopores spontaneously and with remarkable affinity span the lipid bilayer formed over the planar bio-protonic electrode surface and mediate proton transport across the bilayer. Using the ability to easily modify DNA nanostructures, we illustrate that this bioprotonic device can be programmed for electronic recognition of biomolecular signals such as presence of Streptavidin and the cardiac biomarker B-type natriuretic peptide, without modifying the biomolecules. We anticipate this robust interface will allow facile electronic measurement and quantification of biomolecules in a multiplexed manner.

DNA-Au (111) interactions and transverse charge transport properties for DNA-based electronic devices.

DNA's charge transfer and self-assembly characteristics have made it a hallmark of molecular electronics for the past two decades. A fast and efficient charge transfer mechanism with programmable properties using DNA nanostructures is required for DNA-based nanoelectronic applications and devices. The ability to integrate DNA with inorganic substrates becomes critical in this process. Such integrations may affect the conformation of DNA, altering its charge transport properties. Thus, using molecular dynamics simulations and first-principles calculations in conjunction with Green's function approach, we explore the impact of the Au (111) substrate on the conformation of DNA and analyze its effect on the charge transport. Our results indicate that DNA sequence, leading to its molecular conformation on the Au substrate, is critical to engineer charge transport properties. We demonstrate that DNA fluctuates on a gold substrate, sampling various distinct conformations over time. The energy levels, spatial locations of molecular orbitals and the DNA/Au contact atoms can differ between these distinct conformations. Depending on the sequence, at the HOMO, the charge transmission differs up to 60 times between the top ten conformations. We demonstrate that the relative positions of the nucleobases are critical in determining the conformations and the coupling between orbitals. We anticipate that these results can be extended to other inorganic surfaces and pave the way for understanding DNA-inorganic interface interactions for future DNA-based electronic device applications.

Computational study of the role of counterions and solvent dielectric in determining the conductance of B-DNA.

DNA naturally exists in a solvent environment, comprising water and salt molecules such as sodium, potassium, magnesium, etc. Along with the sequence, the solvent conditions become a vital factor determining DNA structure and thus its conductance. Over the last two decades, researchers have measured DNA conductivity both in hydrated and almost dry (dehydrated) conditions. However, due to experimental limitations (the precise control of the environment), it is very difficult to analyze the conductance results in terms of individual contributions of the environment. Therefore, modeling studies can help us to gain a valuable understanding of various factors playing a role in charge transport phenomena. DNA naturally has negative charges located at the phosphate groups in the backbone, which provides both the connections between the base pairs and the structural support for the double helix. Positively charged ions such as the sodium ion (Na^{+}), one of the most commonly used counterions, balance the negative charges at the backbone. This modeling study investigates the role of counterions both with and without the solvent (water) environment in charge transport through double-stranded DNA. Our computational experiments show that in dry DNA, the presence of counterions affects electron transmission at the lowest unoccupied molecular orbital energies. However, in solution, the counterions have a negligible role in transmission. Using the polarizable continuum model calculations, we demonstrate that the transmission is significantly higher at both the highest occupied and lowest unoccupied molecular orbital energies in a water environment as opposed to in a dry one. Moreover, calculations also show that the energy levels of neighboring bases are more closely aligned to ease electron flow in the solution.

Mapping DNA Conformations Using Single-Molecule Conductance Measurements.

DNA is an attractive material for a range of applications in nanoscience and nanotechnology, and it has recently been demonstrated that the electronic properties of DNA are uniquely sensitive to its sequence and structure, opening new opportunities for the development of electronic DNA biosensors. In this report, we examine the origin of multiple conductance peaks that can occur during single-molecule break-junction (SMBJ)-based conductance measurements on DNA. We demonstrate that these peaks originate from the presence of multiple DNA conformations within the solutions, in particular, double-stranded B-form DNA (dsDNA) and G-quadruplex structures. Using a combination of circular dichroism (CD) spectroscopy, computational approaches, sequence and environmental controls, and single-molecule conductance measurements, we disentangle the conductance information and demonstrate that specific conductance values come from specific conformations of the DNA and that the occurrence of these peaks can be controlled by controlling the local environment. In addition, we demonstrate that conductance measurements are uniquely sensitive to identifying these conformations in solutions and that multiple configurations can be detected in solutions over an extremely large concentration range, opening new possibilities for examining low-probability DNA conformations in solutions.

Role of intercalation in the electrical properties of nucleic acids for use in molecular electronics.

Intercalating ds-DNA/RNA with small molecules can play an essential role in controlling the electron transmission probability for molecular electronics applications such as biosensors, single-molecule transistors, and data storage. However, its applications are limited due to a lack of understanding of the nature of intercalation and electron transport mechanisms. We addressed this long-standing problem by studying the effect of intercalation on both the molecular structure and charge transport along the nucleic acids using molecular dynamics simulations and first-principles calculations coupled with the Green's function method, respectively. The study on anthraquinone and anthraquinone-neomycin conjugate intercalation into short nucleic acids reveals some universal features: (1) the intercalation affects the transmission by two mechanisms: (a) inducing energy levels within the bandgap and (b) shifting the location of the Fermi energy with respect to the molecular orbitals of the nucleic acid, (2) the effect of intercalation was found to be dependent on the redox state of the intercalator: while oxidized anthraquinone decreases, reduced anthraquinone increases the conductance, and (3) the sequence of the intercalated nucleic acid further affects the transmission: lowering the AT-region length was found to enhance the electronic coupling of the intercalator with GC bases, hence yielding an increase of more than four times in conductance. We anticipate our study to inspire designing intercalator-nucleic acid complexes for potential use in molecular electronics via creating a multi-level gating effect.

Detection and identification of genetic material via single-molecule conductance.

The ongoing discoveries of RNA modalities (for example, non-coding, micro and enhancer) have resulted in an increased desire for detecting, sequencing and identifying RNA segments for applications in food safety, water and environmental protection, plant and animal pathology, clinical diagnosis and research, and bio-security. Here, we demonstrate that single-molecule conductance techniques can be used to extract biologically relevant information from short RNA oligonucleotides, that these measurements are sensitive to attomolar target concentrations, that they are capable of being multiplexed, and that they can detect targets of interest in the presence of other, possibly interfering, RNA sequences. We also demonstrate that the charge transport properties of RNA:DNA hybrids are sensitive to single-nucleotide polymorphisms, thus enabling differentiation between specific serotypes of Escherichia coli. Using a combination of spectroscopic and computational approaches, we determine that the conductance sensitivity primarily arises from the effects that the mutations have on the conformational structure of the molecules, rather than from the direct chemical substitutions. We believe that this approach can be further developed to make an electrically based sensor for diagnostic purposes.

Morphological Versatility in the Self-Assembly of Val-Ala and Ala-Val Dipeptides.

Since the discovery of dipeptide self-assembly, diphenylalanine (Phe-Phe)-based dipeptides have been widely investigated in a variety of fields. Although various supramolecular Phe-Phe-based structures including tubes, vesicles, fibrils, sheets, necklaces, flakes, ribbons, and wires have been demonstrated by manipulating the external physical or chemical conditions applied, studies of the morphological diversity of dipeptides other than Phe-Phe are still required to understand both how these small molecules respond to external conditions such as the type of solvent and how the peptide sequence affects self-assembly and the corresponding molecular structures. In this work, we investigated the self-assembly of valine-alanine (Val-Ala) and alanine-valine (Ala-Val) dipeptides by varying the solvent medium. It was observed that Val-Ala dipeptide molecules may generate unique self-assembly-based morphologies in response to the solvent medium used. Interestingly, when Ala-Val dipeptides were utilized as a peptide source instead of Val-Ala, we observed distinct differences in the final dipeptide structures. We believe that such manipulation may not only provide us with a better understanding of the fundamentals of the dipeptide self-assembly process but also may enable us to generate novel peptide-based materials for various applications.

Single-step fabrication of patterned gold film array by an engineered multi-functional peptide.

This study constitutes a demonstration of the biological route to controlled nano-fabrication via modular multi-functional inorganic-binding peptides. Specifically, we use gold- and silica-binding peptide sequences, fused into a single molecule via a structural peptide spacer, to assemble pre-synthesized gold nanoparticles on silica surface, as well as to synthesize nanometallic particles in situ on the peptide-patterned regions. The resulting film-like gold nanoparticle arrays with controlled spatial organization are characterized by various microscopy and spectroscopy techniques. The described bio-enabled, single-step synthetic process offers many advantages over conventional approaches for surface modifications, self-assembly and device fabrication due to the peptides' modularity, inherent biocompatibility, material specificity and catalytic activity in aqueous environments. Our results showcase the potential of artificially-derived peptides to play a key role in simplifying the assembly and synthesis of multi-material nano-systems in environmentally benign processes.

Probing the molecular mechanisms of quartz-binding peptides.

Understanding the mechanisms of biomineralization and the realization of biology-inspired inorganic materials formation largely depends on our ability to manipulate peptide/solid interfacial interactions. Material interfaces and biointerfaces are critical sites for bioinorganic synthesis, surface diffusion, and molecular recognition. Recently adapted biocombinatorial techniques permit the isolation of peptides recognizing inorganic solids that are used as molecular building blocks, for example, as synthesizers, linkers, and assemblers. Despite their ubiquitous utility in nanotechnology, biotechnology, and medicine, the fundamental mechanisms of molecular recognition of engineered peptides binding to inorganic surfaces remain largely unknown. To explore propensity rules connecting sequence, structure, and function that play key roles in peptide/solid interactions, we combine two different approaches: a statistical analysis that searches for highly enriched motifs among de novo designed peptides, and, atomistic simulations of three experimentally validated peptides. The two strong and one weak quartz-binding peptides were chosen for the simulations at the quartz (100) surface under aqueous conditions. Solution-based peptide structures were analyzed by circular dichroism measurements. Small and hydrophobic residues, such as Pro, play a key role at the interface by making close contact with the solid and hindering formation of intrapeptide hydrogen bonds. The high binding affinity of a peptide may be driven by a combination of favorable enthalpic and entropic effects, that is, a strong binder may possess a large number of possible binding configurations, many of which having relatively high binding energies. The results signify the role of the local molecular environment among the critical residues that participate in solid binding. The work herein describes molecular conformations inherent in material-specific peptides and provides fundamental insight into the atomistic understanding of peptide/solid interfaces.

Molecular recognition and supramolecular self-assembly of a genetically engineered gold binding peptide on Au{111}.

The understanding of biomineralization and realization of biology-inspired materials technologies depends on understanding the nature of the chemical and physical interactions between proteins and biominerals or synthetically made inorganic materials. Recently, combinatorial genetic techniques permit the isolation of peptides recognizing specific inorganic materials that are used as molecular building blocks for novel applications. Little is known about the molecular structure of these peptides and the specific recognition mechanisms onto their counterpart inorganic surfaces. Here, we report high-resolution atomic force microscopy (AFM), molecular simulation (MS), and geometrical docking studies that detail the formation of an ordered supramolecular self-assembly of a genetically engineered gold binding peptide, 3rGBP(1) ([MHGKTQATSGTIQS](3)), correlating with the symmetry of the Au{111} surface lattice. Using simulated annealing molecular dynamics (SA/MD) studies based on nuclear magnetic resonance (NMR), we confirmed the intrinsic disorder of 3rGBP(1) and identified putative Au docking sites where surface-exposed side chains align with both the <110> and <211> Miller indices of the Au lattice. Our results provide fundamental insight for an atomistic understanding of peptide/solid interfaces and the intrinsic disorder that is inherent in some of these peptide sequences. Analogous to the well-established atomically controlled thin-film heterostructure formation on semiconductor substrates, the basis of today's microelectronics, the fundamental observations of peptide-solid interactions here may well form the basis of peptide-based hybrid molecular technologies of the future.

Effect of molecular conformations on the adsorption behavior of gold-binding peptides.

Despite extensive recent reports on combinatorially selected inorganic-binding peptides and their bionanotechnological utility as synthesizers and molecular linkers, there is still only limited knowledge about the molecular mechanisms of peptide binding to solid surfaces. There is, therefore, much work that needs to be carried out in terms of both the fundamentals of solid-binding kinetics of peptides and the effects of peptide primary and secondary structures on their recognition and binding to solid materials. Here we discuss the effects of constraints imposed on FliTrx-selected gold-binding peptide molecular structures upon their quantitative gold-binding affinity. We first selected two novel gold-binding peptide (AuBP) sequences using a FliTrx random peptide display library. These were, then, synthesized in two different forms: cyclic (c), reproducing the original FliTrx gold-binding sequence as displayed on bacterial cells, and linear (l) dodecapeptide gold-binding sequences. All four gold-binding peptides were then analyzed for their adsorption behavior using surface plasmon resonance spectroscopy. The peptides exhibit a range of binding affinities to and adsorption kinetics on gold surfaces, with the equilibrium constant, Keq, varying from 2.5x10(6) to 13.5x10(6) M(-1). Both circular dichroism and molecular mechanics/energy minimization studies reveal that each of the four peptides has various degrees of random coil and polyproline type II molecular conformations in solution. We found that AuBP1 retained its molecular conformation in both the c- and l-forms, and this is reflected in having similar adsorption behavior. On the other hand, the c- and l-forms of AuBP2 have different molecular structures, leading to differences in their gold-binding affinities.

A novel knowledge-based approach to design inorganic-binding peptides.

MOTIVATION: The discovery of solid-binding peptide sequences is accelerating along with their practical applications in biotechnology and materials sciences. A better understanding of the relationships between the peptide sequences and their binding affinities or specificities will enable further design of novel peptides with selected properties of interest both in engineering and medicine. RESULTS: A bioinformatics approach was developed to classify peptides selected by in vivo techniques according to their inorganic solid-binding properties. Our approach performs all-against-all comparisons of experimentally selected peptides with short amino acid sequences that were categorized for their binding affinity and scores the alignments using sequence similarity scoring matrices. We generated novel scoring matrices that optimize the similarities within the strong-binding peptide sequences and the differences between the strong- and weak-binding peptide sequences. Using the scoring matrices thus generated, a given peptide is classified based on the sequence similarity to a set of experimentally selected peptides. We demonstrate the new approach by classifying experimentally characterized quartz-binding peptides and computationally designing new sequences with specific affinities. Experimental verifications of binding of these computationally designed peptides confirm our predictions with high accuracy. We further show that our approach is a general one and can be used to design new sequences that bind to a given inorganic solid with predictable and enhanced affinity.

Adsorption behavior of linear and cyclic genetically engineered platinum binding peptides.

Recently, phage and cell-surface display libraries have been adapted for genetically selecting short peptides for a variety of inorganic materials. Despite the enormous number of inorganic-binding peptides reported and their bionanotechnological utility as synthesizers and molecular linkers, there is still a limited understanding of molecular mechanisms of peptide recognition of and binding to solid materials. As part of our goal of genetically designing these peptides, understanding the binding kinetics and thermodynamics, and using the peptides as molecular erectors, in this report we discuss molecular structural constraints imposed upon the quantitative binding characteristics of peptides with an affinity for inorganics. Specifically, we use a high-affinity seven amino acid Pt-binding sequence, PTSTGQA, as we reported in earlier studies and build two constructs: one is a Cys-Cys constrained "loop" sequence (CPTSTGQAC) that mimics the domain used in the pIII tail sequence of the phage library construction, and the second is the linear form, a septapeptide, without the loop. Both sequences were analyzed for their adsorption behavior on Pt thin films by surface plasmon resonance (SPR) spectroscopy and for their conformational properties by circular dichroism (CD). We find that the cyclic peptide of the integral Pt-binding sequence possesses single or 1:1 Langmuir adsorption behavior and displays equilibrium and adsorption rate constants that are significantly larger than those obtained for the linear form. Conversely, the linear form exhibits biexponential Langmuir isotherm behavior with slower and weaker binding. Furthermore, the structure of the cyclic version was found to adopt a random coil molecular conformation, whereas the linear version adopts a polyproline type II conformation in equilibrium with the random coil. The 2,2,2-trifluoroethanol titration experiments indicate that TFE has a different effect on the secondary structures of the linear and cyclic versions of the Pt binding sequence. We conclude that the presence of the Cys-Cys restraint affects both the conformation and binding behavior of the integral Pt-binding septapeptide sequence and that the presence or absence of constraints could be used to tune the adsorption and structural features of inorganic binding peptide sequences.

Materials specificity and directed assembly of a gold-binding peptide.

Adsorption studies of a genetically engineered gold-binding peptide, GBP1, were carried out using a quartz-crystal microbalance (QCM) to quantify its molecular affinity to noble metals. The peptide showed higher adsorption onto and lower desorption from a gold surface compared to a platinum substrate. The material specificity, that is, the preferential adsorption, of GBP1 was also demonstrated using gold and platinum micropatterned on a silicon wafer containing native oxide. The biotinylated three-repeat units of GBP1 were preferentially adsorbed onto gold regions delineated using streptavidin-conjugated quantum dots (SAQDs). These experiments not only demonstrate that an inorganic-binding peptide could preferentially adsorb onto a metal (Au) rather than an oxide (SiO2) but also onto one noble metal (Au) over another (Pt). This result shows the utility of an engineered peptide as a molecular erector in the directed immobilization of a nanoscale hybrid entity (SAQDs) over selected regions (Au) on a fairly complex substrate (Au and Pt micropatterned regions on silica). The selective and controlled adsorption of inorganic-binding peptides may have significant implications in nano- and nanobiotechnology, where they could be genetically tailored for specific use in the development of self-assembled molecular systems.

Adsorption kinetics of an engineered gold binding Peptide by surface plasmon resonance spectroscopy and a quartz crystal microbalance.

The adsorption kinetics of an engineered gold binding peptide on gold surface was studied by using both quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) spectroscopy systems. The gold binding peptide was originally selected as a 14-amino acid sequence by cell surface display and then engineered to have a 3-repeat form (3R-GBP1) with improved binding characteristics. Both sets of adsorption data for 3R-GBP1 were fit to Langmuir models to extract kinetics and thermodynamics parameters. In SPR, the adsorption onto the surface shows a biexponential behavior and this is explained as the effect of bimodal surface topology of the polycrystalline gold substrate on 3R-GBP1 binding. Depending on the concentration of the peptide, a preferential adsorption on the surface takes place with different energy levels. The kinetic parameters (e.g., K(eq) approximately 10(7) M(-1)) and the binding energy (approximately -8.0 kcal/mol) are comparable to synthetic-based self-assembled monolayers. The results demonstrate the potential utilization of genetically engineered inorganic surface-specific peptides as molecular substrates due to their binding specificity, stability, and functionality in an aqueous-based environment.

Metal recognition of septapeptides via polypod molecular architecture.

The understanding of the nature of recognition of inorganic materials by proteins is one of the core elements of and has profound implications in biological materials science and engineering. Using combinatorial display methods, a considerable number of short polypeptides have recently been selected with affinity to engineering materials. During these selections, more than several polypeptides are identified with binding specificity to a chosen inorganic material. Understanding the nature of surface recognition of materials by polypeptides is essential for rational design and biomimetic engineering of these inorganic-binding polypeptides for use as linkers, catalyzers, and growth modifiers in nanotechnology and nanobiotechnology. Although there may not be direct homology among the amino acids constituting the polypeptides, their function may come from conserved molecular architecture. Here we study crystallographic surface recognition of platinum metal-binding septapeptides by conformational analysis. We find that the septapeptides conform into certain molecular architectures containing multiple protrusions (polypods) that spatially match with the crystallographic metal surfaces. While the physical recognition may originate from how well the molecular polypods spatially match a given crystallographic surface, the degree of binding may be due to the reactive groups that form the polypods, e.g., charged or polar groups (e.g., hydroxyl and amine). These results are highly consistent with the experimental binding characteristics of the Pt binders with various degrees of affinities.