Enhancing enzyme performance without protein engineering remains a central challenge in biocatalysis. Here, we report the unusual discovery that nanoparticles densely functionalized with DNA, commonly known as spherical nucleic acids (SNAs), can bind enzymes without chemical conjugation, affinity tags, or engineered recognition motifs and substantially increase catalytic activity. Using cytochrome c as a model peroxidase, we show that noncovalent binding to SNAs increases the apparent turnover number by up to 15-fold. Mechanistic studies by varying DNA density, length, and nanoparticle core identity reveal that activity enhancement does not arise from free DNA or the nanoparticle core alone but from the three-dimensional, high-density presentation of DNA at the nanoparticle surface. Consequently, similar effects are observed when gold cores are replaced with glucose oxidase or antibody cores. Moreover, activity modulation also depends on DNA sequence, establishing sequence design as a programmable handle for tuning enzyme output. All-atom molecular dynamics simulations, binding studies, and circular dichroism further reveal that base-specific interactions reshape enzyme conformational flexibility and increase active-site accessibility. This effect generalizes beyond cytochrome c to catalase and carbonic anhydrase. Together, these findings establish SNAs as adaptable, sequence-programmable platforms for enhancing and coordinating biocatalysis and expand their functional scope into enzyme regulation.

Devleena Samanta is an assistant professor of chemistry at The University of Texas at Austin (UT Austin), an associate member of the UT Austin Cancer Research Institute, and a member of the Texas Materials Institute. She received her Ph.D. from Stanford University and completed postdoctoral training at Northwestern University. Her laboratory develops nanoscale tools to sense and program biomolecular function, with applications in diagnostics, therapeutics, and biocatalysis. She is a 2024 Packard Fellow for Science and Engineering and a 2026 Cottrell Scholar.

Enzymes interfaced with nanomaterials often lose more than 90% of their activity; yet, some nanomaterials have been shown to enhance enzyme activity. However, these findings are largely observational and lack clear and actionable design principles. Systematic studies are needed to develop nanomaterials that can control and tune enzyme activity. Given that enzyme-nanomaterial interactions are mediated by their surface functional groups, we hypothesized that engineering nanoparticle surfaces could allow for controlled tuning of the enzyme activity. In this study, we used peptide-functionalized gold nanoparticles (PGNPs) as a programmable platform to investigate how surface functionalization affects enzyme activity. By varying the peptide sequences, we examined the effects of charge, hydrophobicity, peptide length, and structure on the peroxidase activity of cytochrome C (Cyt C). Our results showed that carefully designed ligands can significantly enhance enzyme activity, exceeding 10-fold compared with the free enzyme. Molecular dynamics simulations provided insights into the molecular basis of these findings, revealing the preferred orientation of Cyt C upon adsorption and key interaction patterns between the enzyme and peptide ligands, thus bridging experimental results with a mechanistic understanding. Furthermore, PGNPs proved to be a versatile platform for boosting peroxidase activity of other heme-containing proteins such as lactoperoxidase, hemoglobin, and catalase by 13.4-, 3.9-, and 4.2-fold, respectively. This study highlights the potential of nanoparticle surface engineering to activate enzymes at interfaces in a tunable manner, offering a promising alternative to protein engineering for developing biocatalysts.

Engineering allosteric control sites into enzymes typically requires extensive protein modification. Here, we introduce single-molecule DNA tweezers (SMDTs), which enable programmable, allosteric-like regulation of enzyme activity in response to user-defined chemical cues, without altering the enzyme itself. SMDTs consist of two aptamers connected by a tunable, stimuli-responsive DNA linker. By binding non-covalently to two distinct sites on an enzyme, the SMDT adopts a "pinched" conformation, reminiscent of mechanical tweezers, that inhibits enzymatic activity. Upon exposure to specific molecular triggers, the SMDT undergoes a conformational change that releases the inhibitory aptamer, restoring function. The degree of inhibition and reactivation efficiency can be finely tuned by adjusting the DNA linker's length, sequence, flexibility, and geometry. Operating at nanomolar concentrations, the system exhibits high specificity, capable of discriminating between closely related inputs, including single-base mismatches in nucleic acids. Importantly, SMDTs can be programmed to respond not only to molecular abundance but also to molecular activity. We show the versatility of this platform by regulating enzymes using diverse triggers, including nucleic acids, transcription factors (TATA-binding protein [TBP], cellular myelocytomatosis [c-Myc]), signaling proteins (platelet-derived growth factor [PDGF]), small molecules (kanamycin), and metal ions (Mn2+). These results establish a generalizable framework for designing responsive protein binders that translate molecular recognition into functional outcomes.

Nanoparticles (NPs) are known to enhance the activity of enzymes, but such findings remain largely empirical, lacking predictive design principles. Here, we introduce the first high-throughput platform for the discovery of surface-engineered nanoparticles (SENs) that modulate enzyme function. Guided by the hypothesis that surface ligands are primary drivers of activity enhancement, we synthesized a library of 194 gold- and palladium-based SENs functionalized with diverse peptide ligands. These SENs were screened against three model enzymes: cytochrome c, lactoperoxidase (LPO), and lipase. Multiple SENs substantially increased enzymatic activity, with the most effective achieving ~19-fold increase. The resulting dataset enabled the training of a machine learning model that identified key ligand features associated with high-performing SENs, establishing a predictive framework for designing activity-enhancing NPs. Mechanistic studies confirmed that the ligand shell plays a dominant role in driving enhancement, suggesting that effective ligands identified via this approach can be readily transferred across NP platforms. To demonstrate functional relevance, we show that an optimized SEN/LPO pair outperforms LPO in inhibiting the growth of multidrug-resistant bacteria and disrupting biofilm formation. Collectively, this work offers a scalable and generalizable method to map and harness nanoscale structure-function relationships at biointerfaces, with applications in biocatalysis, biosensing, and beyond.

Nanoparticles (NPs) are known to enhance the activity of enzymes, but such findings remain largely empirical, lacking predictive design principles. Here, the first high-throughput platform for the discovery of surface-engineered nanoparticles (SENs) that modulate enzyme function is introduced. Guided by the hypothesis that surface ligands are primary drivers of activity enhancement, a library of 194 gold- and palladium-based SENs functionalized with diverse peptide ligands is synthesized. These SENs are screened against three model enzymes: cytochrome c, lactoperoxidase (LPO), and lipase. Multiple SENs substantially increased enzymatic activity, with the most effective achieving ≈19-fold increase. The resulting dataset enabled the training of a machine learning model that identified key ligand features associated with high-performing SENs, establishing a predictive framework for designing activity-enhancing NPs. Mechanistic studies confirm that the ligand shell plays a dominant role in driving enhancement, suggesting that effective ligands identified via this approach can be readily transferred across NP platforms. To demonstrate functional relevance, it is shown that an optimized SEN/LPO pair outperforms LPO in inhibiting the growth of multidrug-resistant bacteria and disrupting biofilm formation. Collectively, this work offers a scalable and generalizable method to map and harness nanoscale structure-function relationships at biointerfaces, with applications in biocatalysis, biosensing, and beyond.

The accurate determination of ligand density on DNA-functionalized nanoparticles (DNPs) is critical for biosensing and therapeutic applications, as DNA density directly affects target binding, cellular uptake, and therapeutic efficacy. Traditional methods involve centrifugation to separate unbound DNA from DNPs, followed by DNA quantification to calculate the number of strands per particle. However, this approach is ineffective for centrifugation-resistant DNPs. Alternative methods, such as centrifugal filtration, require multiple washing steps, often resulting in sample loss, and can be labor- and time-intensive, taking several hours. Additionally, many existing techniques rely on expensive labels or toxic reagents. Here, we present a simple, rapid, and label-free method for determining DNA loading using diethylaminoethyl (DEAE)-functionalized beads. We demonstrate its effectiveness on various DNPs, including those with metallic or protein-based cores and various DNA lengths, sequences, and densities. This approach exploits the dif-ferent binding strengths of free and nanoparticle-bound DNA to DEAE, allowing the selective elution of free DNA by adjusting the ionic strength. The eluted DNA is quantified to determine the unbound fraction, which is then used to calculate the number of bound DNA strands per nanoparticle. Requiring only stand-ard laboratory equipment—such as a benchtop centrifuge, a shaker, and either a UV-Vis spectrophotome-ter or a commonly available plate reader—this method provides a fast and reliable alternative to conven-tional approaches, delivering results within 5 minutes. Its versatility and broad applicability across diverse DNP core materials and sizes make it a valuable tool for DNA quantification in a wide range of nanoparti-cle-based platforms.

We introduce a new class of chemical probes for activity-based sensing of proteases, termed cleavable, locked initiator probes (CLIPs). CLIPs contain a protease-cleavable peptide linked between two programmable DNA strands-an "initiator" DNA and a shorter "blocking" DNA. These DNA sequences are designed to hybridize, creating a "locked" hairpin-like structure. Upon proteolytic cleavage, the initiator strand is released, triggering the activation of CRISPR-Cas12a enzymes and producing an amplified fluorescence response. CLIPs generate more than 20-fold turn-on signals at room temperature (25 °C), significantly outperforming commercial probes by yielding ∼40-fold lower limits of detection (LOD) at 100-fold lower concentrations. Their versatility enables the detection of various disease-relevant proteases-including the SARS-CoV-2 main protease, caspase-3, matrix metalloproteinase-7, and cathepsin B-simply by altering the peptide sequence. Importantly, CLIPs detect cathepsin B in four different colorectal cancer cell lines, highlighting their clinical potential. Taken together, the sensitivity (LOD: ∼88 pM), selectivity, and rapid assay time (down to 35 min), combined with the ability to operate in complex biological media with minimal sample preparation, position CLIPs as powerful chemical tools for activity-based sensing of functional enzymes.

Enhancing enzyme functionality while retaining stability has been a long-standing challenge in chemistry. In this issue of Chem, Tan and co-workers present a strategy for encasing enzymes within DNA nanostructures, effectively addressing this limitation. They demonstrate the broad utility of this approach in catalysis, chemical sensing, and tumor therapy.

The accurate determination of DNA loading on spherical nucleic acids (SNAs) is critical for biosensing and therapeutic applications, as DNA loading directly affects target binding, cellular uptake, and therapeutic efficacy. Traditional methods involve centrifugation to separate unbound DNA from SNAs, followed by DNA quantification to calculate the number of strands per particle. However, this approach is ineffective for centrifugation-resistant SNAs. Alternative methods, such as centrifugal filtration, require multiple washing steps, often resulting in sample loss, and can be labor- and time-intensive, taking several hours. Additionally, many existing techniques rely on expensive labels or toxic reagents. Here, we present a simple, rapid, and label-free method for determining DNA loading using diethylaminoethyl (DEAE)-functionalized beads. We demonstrate its effectiveness on centrifugation-resistant SNAs, including those with metallic or protein-based cores and various DNA lengths, sequences, and densities. This approach exploits the different binding strengths of free and nanoparticle-bound DNA to DEAE, allowing the selective elution of free DNA by adjusting the ionic strength. The eluted DNA is quantified to determine the unbound fraction, which is then used to calculate the number of bound DNA strands per nanoparticle. Requiring only standard laboratory equipment─such as a benchtop centrifuge, a shaker, and either a UV-vis spectrophotometer or a commonly available plate reader─this method provides a fast and reliable alternative to conventional approaches, delivering results in under 15 min. Its versatility and broad applicability across diverse SNA core materials and sizes make it a valuable tool for DNA quantification in a wide range of nanoparticle-based platforms.

Oligonucleotide therapeutics are revolutionizing disease treatment by regulating molecules at the genetic level, offering the possibility of treating conditions that were once considered ‘undruggable’. However, delivering oligonucleotides to tissues beyond the liver remains a key challenge, limiting their clinical applications thus far to niche indications. To achieve broader applicability, extensive biomolecular engineering is necessary to enhance the stability, tissue targetability, pharmacokinetics and pharmacodynamics of these structures. The intricate design of these molecules also demands sophisticated process-engineering techniques. Here we provide a collaborative Perspective from academia and industry on the pivotal role of chemical engineering in expanding the use of therapeutic oligonucleotides to treat a wider range of diseases. We discuss how the interplay between biomolecular and process engineering impacts the developability of next-generation oligonucleotide therapeutics as well as their translation from bench to bedside. Oligonucleotide therapeutics have emerged as a promising alternative to traditional small-molecule and protein-based drugs. This Perspective discusses how chemical engineering can broaden oligonucleotide applications to extrahepatic diseases and enable larger-scale production, ultimately allowing treatment of more prevalent conditions than is currently possible.

Nanopore sensing has been successfully used to characterize biological molecules with single-molecule resolution based on the resistive pulse sensing approach. However, its use in nanoparticle characterization has been constrained by the need to tailor the nanopore aperture size to the size of the analyte, precluding the analysis of heterogeneous samples. Additionally, nanopore sensors often require the use of high salt concentrations to improve the signal-to-noise ratio, which further limits their ability to study a wide range of nanoparticles that are unstable at high ionic strength. Here, a new paradigm in nanopore research that takes advantage of a polymer electrolyte system to comprise a conductive pulse sensing approach is presented. A finite element model is developed to explain the conductive pulse signals observed and compare these results with experiments. This system enables the analytical characterization of heterogeneous nanoparticle mixtures at low ionic strength . Furthermore, the wide applicability of the method is demonstrated by characterizing metallic nanospheres of varied sizes, plasmonic nanostars with various degrees of branching, and protein-based spherical nucleic acids with different oligonucleotide loadings. This system will complement the toolbox of nanomaterials characterization techniques to enable real-time optimization workflow for engineering a wide range of nanomaterials.

Enzymes serve as pivotal components in various biotechnological applications across several industries. Understanding enzyme inhibition sheds light on how certain compounds disrupt biochemical pathways, facilitating the design of targeted drugs for combating diseases. On the other hand, reversible inhibition or enhancement of activity can unlock new ways of controlling industrial reactions and boosting the catalytic activity of native enzymes that are taken out of their natural environments. Over the last two decades, immobilizing enzymes on nanomaterial-based solid supports has emerged as an especially promising approach for tuning enzyme activity. Nanomaterials not only inhibit enzymes but also enhance their performance, showcasing their versatility. This Concept highlights significant advancements in utilizing nanomaterials for enzyme modulation and discusses future prospects for leveraging this phenomenon in developing sophisticated molecular systems and downstream applications.

Oligonucleotide therapeutics are revolutionizing disease treatment by regulating molecules at the genetic level, offering the possibility of treating conditions that were once considered "undruggable." However, delivering oligonucleotides to tissues beyond the liver remains a key challenge, limiting their clinical applications thus far to niche indications. To achieve broader applicability, extensive biomolecular engineering is necessary to enhance the stability, tissue targetability, pharmacokinetics, and pharmacodynamics of these structures. The intricate design of these molecules also demands sophisticated process engineering techniques. Herein, we provide a collaborative Perspective from academia and industry on the pivotal role of chemical engineering in expanding the use of therapeutic oligonucleotides to treat a wider range of diseases. We discuss how the interplay between biomolecular and process engineering impacts the developability of next-generation oligonucleotide therapeutics as well as their translation from bench to bedside.

The i-motif is a pH-responsive cytosine-rich oligonucleotide sequence that forms, under acidic conditions, a quadruplex structure. This tunable structural switching has made the i-motif a useful platform for designing pH-responsive nanomaterials. Despite the widespread application of i-motif DNA constructs as biomolecular switches, the mechanism of i-motif folding on the atomic scale has yet to be established. We investigate the early folding structural dynamics of i-motif oligonucleotides with laser-pulse-induced pH-jump time-resolved X-ray solution scattering. Following the pH-jump, we observe that the initial random coil ensemble converts into a contracted intermediate state within 113 ns followed by further folding on the 10 ms time scale. We reveal the representative structures of these transient species, hitherto unknown, with molecular dynamics simulations and ensemble fitting. These results pave the way for understanding metastable conformations of i-motif folding and for benchmarking emerging theoretical models for simulating noncanonical nucleic acid structures.

Protein-based therapeutics have led to new paradigms in disease treatment. Projected to be half of the top ten selling drugs in 2023, proteins have emerged as rivaling and, in some cases, superior alternatives to historically used small molecule-based medicines. This review chronicles both well-established and emerging design strategies that have enabled this paradigm shift by transforming protein-based structures that are often prone to denaturation, degradation, and aggregation in vitro and in vivo into highly effective therapeutics. In particular, we discuss strategies for creating structures with increased affinity and targetability, enhanced in vivo stability and pharmacokinetics, improved cell permeability, and reduced amounts of undesired immunogenicity.

In proximity-driven sensing, interactions between a probe and an analyte produce a detectable signal by causing a change in distance of two probe components or signaling moieties. By interfacing such systems with DNA-based nanostructures, platforms that are highly sensitive, specific, and programmable can be designed. In this Perspective, we delineate the advantages of using DNA building blocks in proximity-driven nanosensors and provide an overview of recent progress in the field, from sensors that rapidly detect pesticides in food to probes that identify rare cancer cells in blood. We also discuss current challenges and identify key areas that need further development.

ABSTRACT Nanopore sensing is a technique based on the Coulter principle to analyze and characterize nanoscale materials with single entity resolution. However, its use in nanoparticle characterization has been constrained by the need to tailor the nanopore aperture size to the size of the analyte, precluding the analysis of heterogenous samples. Additionally, nanopore sensors often require the use of high salt concentrations to improve the signal-to-noise ratio, which further limits their ability to study a wide range of nanoparticles that are unstable at high ionic strength. Here, we report the development of nanopore sensors enhanced by a polymer electrolyte system, enabling the analysis of heterogenous nanoparticle mixtures at low ionic strength. We present a finite element model to explain the anomalous conductive/resistive pulse signals observed and compare these results with experiments. Furthermore, we demonstrate the wide applicability of the method by characterizing metallic nanospheres of varied sizes, plasmonic nanostars with various degrees of branching, and protein-based spherical nucleic acids with different oligonucleotide loadings. Our system will complement the toolbox of nanomaterials characterization techniques and will enable real-time optimization workflow for engineering a wide range of nanomaterials.

CRISPR/Cas systems have revolutionized biology and medicine, and have led to new paradigms in disease diagnostics and therapeutics. However, these complexes suffer from key limitations regarding barriers to cellular entry, stability in biological environments, and off-target effects. Integrating nanotechnology with CRISPR/Cas systems has emerged as a promising strategy to overcome these challenges and has further unlocked structures that accumulate preferentially in tissues of interest, have tunable pharmacological properties, and are activated in response to desired stimuli. Nanomaterials can also enhance CRISPR/Cas-mediated detection platforms by enabling faster, more sensitive, and convenient readouts. We highlight recent advances in this rapidly growing field. We also outline areas that need further development to fully realize the potential of CRISPR technologies.

Patterning biomolecules in synthetic hydrogels offers routes to visualize and learn how spatially-encoded cues modulate cell behavior (e.g., proliferation, differentiation, migration, and apoptosis). However, investigating the role of multiple, spatially defined biochemical cues within a single hydrogel matrix remains challenging because of the limited number of orthogonal bioconjugation reactions available for patterning. Herein, a method to pattern multiple oligonucleotide sequences in hydrogels using thiol-yne photochemistry is introduced. Rapid hydrogel photopatterning of hydrogels with micron resolution DNA features (≈1.5 µm) and control over DNA density are achieved over centimeter-scale areas using mask-free digital photolithography. Sequence-specific DNA interactions are then used to reversibly tether biomolecules to patterned regions, demonstrating chemical control over individual patterned domains. Last, localized cell signaling is shown using patterned protein-DNA conjugates to selectively activate cells on patterned areas. Overall, this work introduces a synthetic method to achieve multiplexed micron resolution patterns of biomolecules onto hydrogel scaffolds, providing a platform to study complex spatially-encoded cellular signaling environments.