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Aptamers are a promising class of affinity reagents because signal transduction mechanisms can be built into the reagent, so that they can directly produce a physically measurable output signal upon target binding. However, endowing the signal transduction functionality into an aptamer remains a trial-and-error process that can compromise its affinity or specificity and typically requires knowledge of the ligand binding domain or its structure. In this work, a design architecture that can convert an existing aptamer into a "reversible aptamer switch" whose kinetic and thermodynamic properties can be tuned without a priori knowledge of the ligand binding domain or its structure is described. Finally, by combining these aptamer switches with evanescent-field-based optical detection hardware that minimizes sample autofluorescence, this study demonstrates the first optical biosensor system that can continuously measure multiple biomarkers (dopamine and cortisol) in complex samples (artificial cerebrospinal fluid and undiluted plasma) with second and subsecond-scale time responses at physiologically relevant concentration ranges., (© 2023 Wiley-VCH GmbH.)

We present a generalizable approach for designing biosensors that can continuously detect small-molecule biomarkers in real time and without sample preparation. This is achieved by converting existing antibodies into target-responsive "antibody-switches" that enable continuous optical biosensing. To engineer these switches, antibodies are linked to a molecular competitor through a DNA scaffold, such that competitive target binding induces scaffold switching and fluorescent signaling of changing target concentrations. As a demonstration, we designed antibody-switches that achieve rapid, sample preparation-free sensing of digoxigenin and cortisol in undiluted plasma. We showed that, by substituting the molecular competitor, we can further modulate the sensitivity of our cortisol switch to achieve detection at concentrations spanning 3.3 nanomolar to 3.3 millimolar. Last, we integrated this switch with a fiber optic sensor to achieve continuous sensing of cortisol in a buffer and blood with <5-min time resolution. We believe that this modular sensor design can enable continuous biosensor development for many biomarkers.

Aptamer-based molecular switches that undergo a binding-induced conformational change have proven valuable for a wide range of applications, such as imaging metabolites in cells, targeted drug delivery, and real-time detection of biomolecules. Since conventional aptamer selection methods do not typically produce aptamers with inherent structure-switching functionality, the aptamers must be converted to molecular switches in a post-selection process. Efforts to engineer such aptamer switches often use rational design approaches based on in silico secondary structure predictions. Unfortunately, existing software cannot accurately model three-dimensional oligonucleotide structures or non-canonical base-pairing, limiting the ability to identify appropriate sequence elements for targeted modification. Here, we describe a massively parallel screening-based strategy that enables the conversion of virtually any aptamer into a molecular switch without requiring any prior knowledge of aptamer structure. Using this approach, we generate multiple switches from a previously published ATP aptamer as well as a newly-selected boronic acid base-modified aptamer for glucose, which respectively undergo signal-on and signal-off switching upon binding their molecular targets with second-scale kinetics. Notably, our glucose-responsive switch achieves ~30-fold greater sensitivity than a previously-reported natural DNA-based switch. We believe our approach could offer a generalizable strategy for producing target-specific switches from a wide range of aptamers., (© 2023. The Author(s).)

Emerging severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants have raised concerns about the efficacy of vaccines. The present study aimed to compare the potential of Delta and Omicron variant-specific mRNA vaccines in inducing immune responses. B cell and T cell epitopes and population coverage of spike (S) glycoprotein of the variants were predicted using the Immune Epitope Database. The molecular docking was carried out between the protein and different toll-like receptors, as well as the receptor-binding domain (RBD) protein and angiotensin-converting-enzyme 2 (ACE2) cellular receptor using ClusPro. The molecular simulation was done for each docked RBD-ACE2 using YASARA. The mRNA secondary structure was predicted through the RNAfold. The simulation of immune responses to the mRNA vaccine construct was performed using C-ImmSim. Apart from a few positions, no significant difference was observed in the prediction of S protein B cell and T cell epitopes of these two variants. The lower amounts of Median consensus percentile in the Delta variant in similar positions signify its stronger affinity to major histocompatibility complex (MHC) II binding alleles. Docking of Delta S protein with TLR3, TLR4, and TLR7 and also its RBD with ACE2 showed striking interactions with the lower binding energy than Omicron. In the immune simulation, elevated levels of cytotoxic T lymphocytes, helper T lymphocytes, and memory cells in both the active and resting states and the main regulators of the immune system suggested the capacity of mRNA constructs to elicit robust immune responses against SARS-CoV-2 variants. Considering slight differences in the binding affinity to MHC II binding alleles, activation of TLRs, mRNA secondary structure stability, and concentration of immunoglobulins and cytokines, the Delta variant is suggested for the mRNA vaccine construction. Further studies are being done to prove the efficiency of the design construct.

Enzyme-linked immunosorbent assays (ELISAs) are a cornerstone of modern molecular detection, but the technique still faces notable challenges. One of the biggest problems is discriminating true signal generated by target molecules versus non-specific background. Here, we developed a Single-Molecule Colocalization Assay (SiMCA) that overcomes this problem by employing total internal reflection fluorescence microscopy to quantify target proteins based on the colocalization of fluorescent signal from orthogonally labeled capture and detection antibodies. By specifically counting colocalized signals, we can eliminate the effects of background produced by non-specific binding of detection antibodies. Using TNF-α, we show that SiMCA achieves a three-fold lower limit of detection compared to conventional single-color assays and exhibits consistent performance for assays performed in complex specimens such as serum and blood. Our results help define the pernicious effects of non-specific background in immunoassays and demonstrate the diagnostic gains that can be achieved by eliminating those effects., (© 2022. The Author(s).)

Although RNA and DNA are best known for their capacity to encode biological information, it has become increasingly clear over the past few decades that these biomolecules are also capable of performing other complex functions, such as molecular recognition (e.g., aptamers) and catalysis (e.g., ribozymes). Building on these foundations, researchers have begun to exploit the predictable base-pairing properties of RNA and DNA in order to utilize nucleic acids as functional materials that can undergo a molecular "switching" process, performing complex functions such as signaling or controlled payload release in response to external stimuli including light, pH, ligand-binding and other microenvironmental cues. Although this field is still in its infancy, these efforts offer exciting potential for the development of biologically based "smart materials". Herein, ongoing progress in the use of nucleic acids as an externally controllable switching material is reviewed. The diverse range of mechanisms that can trigger a stimulus response, and strategies for engineering those functionalities into nucleic acid materials are explored. Finally, recent progress is discussed in incorporating aptamer switches into more complex synthetic nucleic acid-based nanostructures and functionalized smart materials., (© 2020 Wiley-VCH GmbH.)

Background and Purpose: Cancer is the leading cause of death in today's world, therefore the efforts to achieve anticancer drugs with higher potency and fewer side effects have always been conducted by researchers in the field of pharmaceutical chemistry.Monastrol, a cytotoxic small molecule, from dihydropyrimidinone scaffold, is an inhibitor of the kinesin-5 protein. So, efforts to identify more derivatives of this molecule have been of interest., Experimental Approach: Some of monastrol's analogs as Eg5 inhibitors with different substitution patterns were analyzed, synthesized, and their cytotoxic effects were evaluated on MCF-7 and HeLa cancerous cells in vitro using the MTT assay. The structure-activity relationship (SAR) was studied in silico by molecular docking., Findings / Results: Among all proposed structures, in ducking study, those with hydrophobic moieties on the C2-N3 region, those with a hydroxyl group on the phenyl on C4 position, and those with a carboxylic group on C5 were the best candidates. In vitro studies, on the other side, emphasized that monastrol still was the most potent derivative. Another finding was the more moderate activity of synthesized compounds on the HeLa cell compared to the MCF-7 cell line. During different challenges for substitution at 5-position, some earlier reports around the dihydropyrimidinone reactions were questioned. It seems that the change at the position 5 is not merely accessible, as earlier reports claimed. Also, we could not achieve any better cell cytotoxicity by the larger group in the thiourea region or position 5; nonetheless, it seems that the introduction of a methylene group at this position could be beneficial., Conclusion and Implications: The initial results of this study were valuable in terms of design and synthesis and will be useful for future investigations., Competing Interests: The authors declare no conflicts of interest for this study., (Copyright: © 2020 Research in Pharmaceutical Sciences.)

DNA nanotechnology relies on the molecular recognition properties of DNA to produce complex architectures through self-assembly. The resulting DNA nanostructures allow scientists to organize functional materials at the nanoscale and have therefore found applications in many domains of materials science over the past several years. These scaffolds have been used to position proteins, nanoparticles, carbon nanotubes, and other nanomaterials with high spatial resolution. In addition to their remarkable performance as frameworks for other species, DNA constructs also possess interesting dynamic properties, which have led to their use in logic circuits, drug delivery vehicles, and molecular walkers. Although DNA nanostructures have become increasingly complex, the development of tools to study them has lagged. Currently, gel electrophoresis, dynamic light scattering, and ensemble fluorescence measurements are widely used to characterize DNA-based assemblies. Unfortunately, ensemble averaging in these methods obscures malformed structures and may mask properties associated with structure, length, and shape in polydisperse samples. While atomic force microscopy allows for the determination of morphology at the single-molecule level, this technique cannot typically be used to assess the dynamic properties of these constructs. To analyze the function of DNA-based devices such as molecular motors and reconfigurable nanostructures in real time, new single-molecule techniques are required. This Account details the work from our laboratories toward developing single-molecule fluorescence (SMF) methodologies for the structural and dynamic characterization of wireframe DNA nanostructures, one at a time. The methods described herein provide us with two separate yet related sets of information: First, we can statically examine the nanostructures one by one to assess their robustness, structural fidelity, and morphology. This is primarily done using two-color stepwise photobleaching, wherein we can examine the subunit stoichiometry of our assemblies before and after various perturbations to the structures. For example, we can introduce length mismatches to cause the nanotube to bend or perform strand displacement reactions to generate single-stranded, flexible analogues of our materials. Second, due to the unmatched spatiotemporal resolution of SMF techniques, we can study the dynamic character of these assemblies by implementing structural changes to the nanotube and monitoring them in real time. With this structural and dynamic information in hand, our groups have additionally developed new tools for the improved construction of DNA nanotubes, inspired by solid-phase DNA synthesis. By assembling the nanotubes in a stepwise manner, highly monodisperse nanostructures of any desired length can be made without a template strand. In this way, unique building blocks can also be added sequence-specifically, allowing for the production of user-defined scaffolds to organize nanoscale materials in three dimensions. This method, in combination with our imaging and analysis protocols, may be extended to assemble and inspect other supramolecular constructs in a controlled manner. Overall, by combining synthesis, characterization, and analysis, these single-molecule techniques hold the potential to advance the study of DNA nanostructures and dynamic DNA-based devices.

Molecular switches that change their conformation upon target binding offer powerful capabilities for biotechnology and synthetic biology. Aptamers are useful as molecular switches because they offer excellent binding properties, undergo reversible folding, and can be engineered into many nanostructures. Unfortunately, the thermodynamic and kinetic properties of the aptamer switches developed to date are intrinsically coupled, such that high temporal resolution can only be achieved at the cost of lower sensitivity or high background. Here, we describe a design strategy that decouples and enables independent control over the thermodynamics and kinetics of aptamer switches. Starting from a single aptamer, we create an array of aptamer switches with effective dissociation constants ranging from 10 μM to 40 mM and binding kinetics ranging from 170 ms to 3 s. Our strategy is broadly applicable to other aptamers, enabling the development of switches suitable for a diverse range of biotechnology applications.

Dynamic wireframe DNA structures have gained significant attention in recent years, with research aimed toward using these architectures for sensing and encapsulation applications. For these assemblies to reach their full potential, however, knowledge of the rates of strand displacement and hybridization on these constructs is required. Herein, we report the use of single-molecule fluorescence methodologies to observe the reversible switching between double- and single-stranded forms of triangular wireframe DNA nanotubes. Specifically, by using fluorescently labeled DNA strands, we were able to monitor changes in intensity over time as we introduced different sequences. This allowed us to extract detailed kinetic information on the strand displacement and hybridization processes. Due to the polymeric nanotube structure, the ability to individually address each of the three sides, and the inherent polydispersity of our samples as a result of the step polymerization by which they are formed, a library of compounds could be studied independently yet simultaneously. Kinetic models relying on mono-exponential decays, multi-exponential decays, or sigmoidal behavior were adjusted to the different constructs to retrieve erasing and refilling kinetics. Correlations were made between the kinetic behavior observed, the site accessibility, the nanotube length, and the structural robustness of wireframe DNA nanostructures, including fully single-stranded analogs. Overall, our results reveal how the length, morphology, and rigidity of the DNA framework modulate the kinetics of strand displacement and hybridization as well as the overall addressability and structural stability of the structures under study.

A wide variety of approaches have become available for the fabrication of nanomaterials with increasing degrees of complexity, precision, and speed while minimizing cost. Their quantitative characterization, however, remains a challenge. Analytical methods to better inspect and validate the structure and composition of large nanoscale objects are required to optimize their applications in diverse technologies. Here, we describe single-molecule fluorescence-based strategies relying on photobleaching and multiple-color co-localization features toward the characterization of supramolecular structures. By optimizing imaging conditions, including surface passivation, excitation power, frame capture rate, fluorophore choice, buffer media, and antifading agents, we have built a robust method by which to dissect the structure of synthetic nanoscale systems. We showcase the use of our methods by retrieving key structural parameters of four DNA nanotube systems differing in their preparation strategy. Our method rapidly and accurately assesses the outcome of synthetic work building nano- and mesoscale architectures, providing a key tool for product studies in nanomaterial synthesis.

DNA nanotubes hold great potential as drug delivery vehicles and as programmable templates for the organization of materials and biomolecules. Existing methods for their construction produce assemblies that are entirely double-stranded and rigid, and thus have limited intrinsic dynamic character, or they rely on chemically modified and ligated DNA structures. Here, we report a simple and efficient synthesis of DNA nanotubes from 11 short unmodified strands, and the study of their dynamic behavior by atomic force microscopy and in situ single molecule fluorescence microscopy. This method allows the programmable introduction of DNA structural changes within the repeat units of the tubes. We generate and study fully double-stranded nanotubes, and convert them to nanotubes with one, two and three single-stranded sides, using strand displacement strategies. The nanotubes can be reversibly switched between these forms without compromising their stability and micron-scale lengths. We then site-specifically introduce DNA strands that shorten two sides of the nanotubes, while keeping the length of the third side. The nanotubes undergo bending with increased length mismatch between their sides, until the distortion is significant enough to shorten them, as measured by AFM and single-molecule fluorescence photobleaching experiments. The method presented here produces dynamic and robust nanotubes that can potentially behave as actuators, and allows their site-specific addressability while using a minimal number of component strands.

DNA nanotubes offer a high aspect ratio and rigidity, attractive attributes for the controlled assembly of hierarchically complex linear arrays. It is highly desirable to control the positioning of rungs along the backbone of the nanotubes, minimize the polydispersity in their manufacture and reduce the building costs. We report here a solid-phase synthesis methodology in which, through a cyclic scheme starting from a 'foundation rung' specifically bound to the surface, distinct rungs can be incorporated in a predetermined manner. Each rung is orthogonally addressable. Using fluorescently tagged rungs, single-molecule fluorescence studies demonstrated the robustness and structural fidelity of the constructs and confirmed the incorporation of the rungs in quantitative yield (>95%) at each step of the cycle. Prototype structures that consisted of up to 20 repeat units, about 450 nm in contour length, were constructed. Combined, the solid-phase synthesis strategy described and its visualization through single-molecule spectroscopy show good promise for the production of custom-made DNA nanotubes.

Here we report spectroscopic studies on the interaction of negatively charged, amphiphilic polyphenylene ethynylene (PPE) polymers with liposomes prepared either from negative, positive or zwitterionic lipids. Emission spectra of PPEs of 7 and 49 average repeat units bearing carboxylate terminated side chains showed that the polymer embeds within positively charged lipids where it exists as free chains. No interaction was observed between PPEs and negatively charged lipids. Here the polymer remained aggregated giving rise to broad emission spectra characteristic of the aggregate species. In zwitterionic lipids, we observed that the majority of the polymer remained aggregated yet a small fraction readily embedded within the membrane. Titration experiments revealed that saturation of zwitterionic lipids with polymer typically occurred at a polymer repeat unit to lipid mole ratio close to 0.05. No further membrane embedding was observed above that point. For liposomes prepared from positively charged lipids, saturation was observed at a PPE repeat unit to lipid mole ratio of ∼0.1 and liposome precipitation was observed above this point. FRET studies showed that precipitation was preceded by lipid mixing and liposome fusion induced by the PPEs. This behavior was prominent for the longer polymer and negligible for the shorter polymer at a repeat unit to lipid mole ratio of 0.05. We postulate that fusion is the consequence of membrane destabilization whereby the longer polymer gives rise to more extensive membrane deformation than the shorter polymer.

DNA nanotubes have great potential as nanoscale scaffolds for the organization of materials and the templation of nanowires and as drug delivery vehicles. Current methods for making DNA nanotubes either rely on a tile-based step-growth polymerization mechanism or use a large number of component strands and long annealing times. Step-growth polymerization gives little control over length, is sensitive to stoichiometry, and is slow to generate long products. Here, we present a design strategy for DNA nanotubes that uses an alternative, more controlled growth mechanism, while using just five unmodified component strands and a long enzymatically produced backbone. These tubes form rapidly at room temperature and have numerous, orthogonal sites available for the programmable incorporation of arrays of cargo along their length. As a proof-of-concept, cyanine dyes were organized into two distinct patterns by inclusion into these DNA nanotubes.

Background: Choice of adiposity measure may be important in the evaluation of relationships between adiposity and risk markers for cardiovascular disease and diabetes., Aim: We explored the strengths of risk marker associations with BMI, a simple measure of adiposity, and with measures provided by skinfold thicknesses and dual energy X-ray absorptiometry (DXA)., Subjects and Methods: We evaluated in three subgroups of white males (n = 156-349), participating in a health screening program, the strengths of relationship between measures of total and regional adiposity and risk markers relating to blood pressure, lipids and lipoproteins, insulin sensitivity, and subclinical inflammation., Results: Independent of age, smoking, alcohol intake, and exercise, the strongest correlations with adiposity measures were seen with serum triglyceride concentrations and indices of insulin sensitivity, with strengths of association showing little difference between BMI and skinfold and DXA measures of total and percent body fat (R = 0.20-0.46, P < 0.01). Significant but weaker associations with adiposity were seen for serum HDL cholesterol and only relatively inconsistent associations with adiposity for total and LDL cholesterol and indices of subclinical inflammation., Conclusions: BMI can account for variation in risk markers in white males as well as more sophisticated measures derived from skinfold thickness measurements or DXA scanning.