Search for: All records

Abstract Modern molecular detection assays such as enzyme‐linked immunosorbent assays (ELISAs) offer excellent sensitivity and specificity, but typically require multiple reagents and extensive sample preparation, limiting their usefulness as rapid diagnostics. A generalizable biosensor platform is introduced that enables single‐step, sample preparation‐free detection of protein analytes with high sensitivity in complex samples. The NanoFluor system employs Janelia Fluor dyes coupled to a nanobody via HaloTag conjugation with a flexible glycine‐serine linker, where the dye undergoes a switch from a non‐fluorescent to a fluorescent state when the coupled nanobody binds to its target. It is demonstrated that the NanoFluor design achieves detection limits as low as picomolar concentrations across diverse protein targets. Molecular dynamics simulations, coupled with quantum mechanics/molecular mechanics computational models, reveal the mechanistic basis for the fluorescence change, and demonstrate the feasibility of multiplexed detection in complex samples including undiluted serum. This versatile, simple biosensor design can prove valuable for point‐of‐care diagnostics and other molecular detection applications. 

Abstract The pituitary adenylate cyclase-activating polypeptide receptor I (PAC1R) represents a highly sought-after therapeutic target for chronic pain, migraine, and post-traumatic stress. As a class B G protein-coupled receptor (GPCR), the PAC1R is highly expressed throughout the neuronal and central nervous system membranes, with the receptor subject to hormone activation and subsequent signal transduction. Despite its desirable indication, small molecule agonists for PAC1R have been notoriously difficult to develop due to competition with PACAP. For this reason, allosteric activation of the receptor has emerged as a promising pathway. To probe potential allosteric sites, herein, we present a study of the receptor in biomimetic concentrations of lipid membranes. Our results reveal that cholesterol recognizes two canonical and two non-canonical binding sites at PAC1R, which may influence critical residues in the transmembrane domain and PAC1R activation. Also, our simulations suggest the glycolipid GM3 interacts with PAC1R in both the extracellular and transmembrane domains. These lipid binding hotspots may hold high potential for advancing our understanding of class B GPCR signaling and the discovery of new molecules targeting PAC1R. Statement of SignificanceThe PAC1R is a highly sought after therapeutic target in the GPCR family. Understanding its natural process signaling is highly interesting in identifying new modes of control and is currently not well established. This work uses coarse-grained molecular dynamics simulations to examine diverse PAC1R models’ interactions with their endogenous lipid bilayer in compositions that match the regions in the brain where the receptor is expressed. Two lipids, cholesterol and GM3, have been previously identified in similar receptors as allosteric modulators and were specifically examined in this study. This work also showcases multiple new lipid binding sites at transmembrane and extracellular sites highly implicated in PAC1R signaling. 

Abstract The formation of higher‐order structures in natural biopolymers, such as polypeptides and nucleic acids, is governed by sequence specificity and monomer chemistry. Although nucleic acids can assemble into programmable nanostructures through base‐pairing interactions, their chemical diversity is limited to four nucleobases. DNA amphiphiles overcome this limitation by introducing orthogonal interactions through non‐nucleosidic modifications. These amphiphiles self‐assemble into diverse morphologies, such as spheres, fibers, or sheets, with closely packed, parallel DNA strands on their exterior. This unusual arrangement can give rise to emergent properties absent in simple DNA strands. Here, we show that the precise sequence of single‐stranded DNA, independent of double helix base‐pairing, can be used to program the self‐assembled morphology of DNA amphiphiles. Remarkably, small sequence variations can drive the formation of nonequilibrium DNA nanotoroids, rather than conventional morphologies. The DNA nanotoroids were formed as on‐pathway structures via a competitive mechanism, only when a toroid‐selective DNA sequence was used. They could be stabilized noncovalently by a small molecule cross‐linker or coassembly with a secondary DNA amphiphile. Molecular dynamics simulations demonstrated the dependence of toroid formation on the structure of the end π‐stacking unit. This work introduces a new class of DNA‐based nanotoroid materials with assembly properties controlled by unique sequences, akin to proteins, for applications in cell delivery, nanofiltration, nanoreactors, and materials templation. 

Abstract The formation of higher‐order structures in natural biopolymers, such as polypeptides and nucleic acids, is governed by sequence specificity and monomer chemistry. Although nucleic acids can assemble into programmable nanostructures through base‐pairing interactions, their chemical diversity is limited to four nucleobases. DNA amphiphiles overcome this limitation by introducing orthogonal interactions through non‐nucleosidic modifications. These amphiphiles self‐assemble into diverse morphologies, such as spheres, fibers, or sheets, with closely packed, parallel DNA strands on their exterior. This unusual arrangement can give rise to emergent properties absent in simple DNA strands. Here, we show that the precise sequence of single‐stranded DNA, independent of double helix base‐pairing, can be used to program the self‐assembled morphology of DNA amphiphiles. Remarkably, small sequence variations can drive the formation of nonequilibrium DNA nanotoroids, rather than conventional morphologies. The DNA nanotoroids were formed as on‐pathway structures via a competitive mechanism, only when a toroid‐selective DNA sequence was used. They could be stabilized noncovalently by a small molecule cross‐linker or coassembly with a secondary DNA amphiphile. Molecular dynamics simulations demonstrated the dependence of toroid formation on the structure of the end π‐stacking unit. This work introduces a new class of DNA‐based nanotoroid materials with assembly properties controlled by unique sequences, akin to proteins, for applications in cell delivery, nanofiltration, nanoreactors, and materials templation. 

Abstract Biopolymers such as nucleic acids and proteins exhibit dynamic backbone folding, wherein site-specific intramolecular interactions determine overall structure. Proteins then hierarchically assemble into supramolecular polymers such as microtubules, that are robust yet dynamic, constantly growing or shortening to adjust to cellular needs. The combination of dynamic, energy-driven folding and growth with structural stiffness and length control is difficult to achieve in synthetic polymer self-assembly. Here we show that highly charged, monodisperse DNA-oligomers assemble via seeded growth into length-controlled supramolecular fibers during heating; when the temperature is lowered, these metastable fibers slowly disassemble. Furthermore, the specific molecular structures of oligomers that promote fiber formation contradict the typical theory of block copolymer self-assembly. Efficient curling and packing of the oligomers – or ‘curlamers’ – determine morphology, rather than hydrophobic to hydrophilic ratio. Addition of a small molecule stabilises the DNA fibers, enabling temporal control of polymer lifetime and underscoring their potential use in nucleic-acid delivery, stimuli-responsive biomaterials, and soft robotics. 

Peptide coassembly offers novel opportunities for designing advanced nanomaterials. This study used coarse-grained molecular dynamics simulations to examine the coassembly of charge-complementary peptides, assessing various ratios and the role of charge and hydrophobicity in their aggregation. We discovered that peptide length, charge, and hydrophobicity significantly influence coassembly behavior, with more hydrophobic peptides exhibiting greater aggregation despite electrostatic repulsion. Beyond the coassembly of two peptides, we also observed that the coassembly of more than two peptides will likely lead to new assembly structures and properties. Our findings underscore the importance of peptide composition and length in tuning the coassembly and the resulting properties, thus facilitating the design of complex peptide nanoparticles for biomedical and biotechnological applications. 

Wireframe DNA nanocages, an important type of DNA nanomaterials, exhibit exceptional programmability for chemical modifications, along with tunable size and shape. Nevertheless, the impact of their conformational fluctuations on cage design has not been thoroughly explored, despite speculation regarding its influence on biomedical applications. This study marks the first systematic examination of the conformational dynamics of prismatic DNA nanocages through molecular modeling and simulation. By comparing four different DNA nanocage topologies, we uncover design parameter combinations and conditions that facilitate access to varying conformational states. We observe the expansion and contraction of these cages across various topologies, hybridization states, and ionic environments (Mg2+/Na+), with their volumes varying from 15% to 150% of the ideal cage volumes. Our results indicate that the dynamics of DNA cages is influenced by the concentrations of Mg2+ and Na+ ions. Additionally, the flexibility of specific DNA strands can be manipulated, thereby altering the cage volume, through the selective hybridization of the cage edges. Ultimately, the conformational dynamics of DNA nanocages are captured in atomic detail. This study offers valuable modeling tools and methodologies to assist future DNA nanocage design endeavors.