Biology subjects

Kim, S.

Publications and source records attributed to Kim, S..

At least 73 records · Page 4Linked to original sources

bioRxiv2026

Myosin activity drives entangled actin networks out-of-equilibrium - a quantitative approach

ATP-driven myosin II activity remodels actin networks and drives cytoskeletal matter out of thermal equilibrium, but how ATP concentration controls these dynamics remains difficult to isolate in vivo. Here, we reconstitute minimal actomyosin networks from purified components and combine passive microrheology with mean back relaxation (MBR) analysis to quantify ATP-dependent nonequilibrium fluctuations. Nonequilibrium activity is strongest at intermediate ATP concentrations (0.2-0.5 mM) and decreases at higher ATP. While single-bead van Hove distributions are approximately Gaussian, pooled distributions display apparent tails caused mainly by bead-to-bead heterogeneity rather than frequent active bursts. MBR, however, reveals clear time-irreversible dynamics by distinguishing restoring relaxation from persistent active motion. Comparing activity with network stiffness suggests a trade-off between ATP-dependent stiffening and myosin-driven remodeling. A minimal active Langevin simulation reproduces the observed MBR phenomenology, supporting a picture in which rare myosin-driven cage rearrangements generate detectable nonequilibrium signatures. These results establish MBR as a sensitive probe of active matter behavior in actomyosin networks. Significance StatementCells operate out of equilibrium, yet the specific role of ATP concentration in driving cytoskeletal activity remains difficult to isolate in vivo. By reconstituting minimal actomyosin networks and applying passive microrheology, we directly quantify how ATP levels modulate out-of-equilibrium fluctuations. The application of mean back relaxation (MBR) analysis thereby provides a clear and broadly accessible measure of broken time-reversal symmetry that surpasses conventional analysis methods. Our results reveal an inverse relationship between ATP concentration and network dynamics, arising from different modes of myosin activity and ATP-dependent network stiffening. This work provides a quantitative framework for linking biochemical energy supply to mechanical activity in reconstituted cytoskeletal systems, offering new insights into cellular self-organization and energy-dependent regulation.

biophysics↗

bioRxiv2026

A Novel Drug Candidate that Selectively Targets the Critical Androgen Receptor-ELK1 Growth Axis in Advanced and Drug-Resistant Prostate Cancer

Androgen receptor (AR)-dependent prostate cancer (PCa) cells require co-activation of ELK1 by AR to activate a critical set of cell cycle and mitosis genes, regardless of hormone - sensitivity. A small molecule antagonist (KCI807) that inhibits AR-dependent growth by selectively binding to AR and blocking its association with ELK1 is limited as a drug by auto-induced metabolism. Using structure-activity data, consistent with modeling a physically mapped KCI807 binding pocket, we developed a new class of compounds with a different core structure comprising 5-Hydroxy-2-(3-hydroxyphenyl)-1-methylquinolin-4(1H)-one (KCI830), with variable N- substituents. The compound with a N-2,2,2-trifluoroethyl substitution (KCI838) was the fastest acting and most potent inhibitor of AR-dependent cell growth and colony formation in PCa model cells, including exclusively AR splice variant-dependent and other enzalutamide-resistant cells, without affecting growth of AR-negative cell lines. Critical tests were conducted to establish that KCI838 recapitulates the previously elucidated mode of action of KCI807. KCI838 selectively inhibited ELK1-dependent vs. androgen response element (ARE)-driven promoter and gene activation by AR. KCI838 blocked AR binding to ELK1 in situ tested by BRET assay. Increasing the total cellular AR by [~]2-fold using ectopic AR expression caused the predicted change in drug dose-response profile for growth, implicating AR as the exclusive target for the activity of KCI838. KCI838s molecular scaffold conferred reduced enzyme induction in primary human hepatocytes and weakened interactions with human UGT1A1 and CYP1A2. In mice bearing an aggressive, enzalutamide-resistant patient-derived PCa tumor xenograft characteristically overexpressing prostatic acid phosphatase, daily bolus injections of a soluble 3phosphate monoester prodrug of KCI838 (KCI838PME) progressively inhibited tumor growth, concomitant with tumor accumulation of active hydrophobic drug, without significant toxicity. Additionally, ALZET osmotic pumps were used to establish proof-of-concept for reversible in vivo anti-tumor activity of KCI838PME administered in a low dose, controlled release mode. The results warrant investigation of KCI838PME in a controlled-release formulation, to treat PCa that is resistant to current AR-targeted therapies while obviating the need for testosterone suppression.

cancer biology↗

bioRxiv2026

rDNA breaks activate dsRNA pattern recognition through sense-antisense transcription

Myriad DNA damaging chemo- and radio- therapies interfere with ribosomal RNA (rRNA) transcription and processing, yet the biological consequences of these phenomena remain unclear. Here we show that aberrant transcripts emanating from rDNA breaks engage double-stranded RNA pattern recognition receptors, melanoma differentiation-associated protein 5 (MDA5) and retinoic acid-inducible gene I (RIG-I) to activate immune signaling. rDNA damage abolishes full-length rRNA synthesis while generating truncated sense-antisense rRNA transcripts, whose accumulation is restrained by ATM and ATR kinase activities. Purification of the endogenous MDA5-filament coupled with sequencing identified complementary sense and antisense rRNA transcripts that terminate and initiate near the break site, respectively. This implicates aberrant rRNA species as a major source of damage induced endogenous ligands for dsRNA pattern recognition and establishes a mechanism by which nucleolar stress is coupled to immune signaling.

cell biology↗

bioRxiv2026

Dynamic partitioning shapes the in vivo organization of the E. coli RNA degradosome

The bacterial RNA degradosome is a central mediator of RNA turnover, yet how its components are organized and regulated in living cells remains unclear. Using live-cell single-molecule imaging, we quantified the spatial distribution and dynamics of the four major Escherichia coli degradosome components: RNase E, RhlB, PNPase, and enolase. These proteins occupied distinct membrane-associated and cytoplasmic pools whose relative abundance varied among proteins and changed with physiological state. In particular, PNPase underwent pronounced redistribution between membrane-associated and cytoplasmic states in response to altered RNA availability and growth conditions, whereas RNase E and RhlB remained largely membrane associated. To determine the functional consequences of this organization, we examined the degradation of lacZ reporter transcripts with different translation initiation strengths. PNPase and RhlB preferentially promoted degradation of weakly translated transcripts, whereas strongly translated transcripts were largely insensitive to their loss. Together, these results reveal that the bacterial RNA degradosome is not a static, uniformly assembled molecular machine. Instead, RNA decay is organized through dynamic intracellular partitioning of degradosome components, linking physiological state and translation status to transcript degradation.

microbiology↗

bioRxiv2026

Rapid in vitro synthesis of DNA templates via Sidewinder for polyadenylated Hantavirus mRNA vaccine candidates

As the recent COVID-19 pandemic illustrated, zoonotic viruses and other pathogens pose a credible threat to public health. Recent advancements in vaccine technology, particularly mRNA vaccines, provide key tools for an effective and swift public health response. Although mRNA vaccines can be developed more quickly than traditional vaccines, fast and accurate construction of DNA templates for these vaccines remains a critical bottleneck. Using our novel DNA assembly technology, Sidewinder, we rapidly designed and built multiple mRNA vaccine candidates to guard against a potential outbreak of Hantavirus (ANDV). We successfully constructed the DNA templates from oligo pools and produced the mRNA for three vaccine candidates in just 2 days after delivery of the synthetic DNA oligos.

biochemistry↗

bioRxiv2026

Regulation of Pacemaker Current in the Sinoatrial Node by Zonula Occludens-1

BACKGROUNDIn addition to lethal ventricular arrhythmias, arrhythmogenic cardiomyopathy (ACM) is associated with conduction abnormalities, bradycardias, and reduced expression of the scaffolding junctional protein zonula occludens-1 (ZO-1). Reduced ZO-1 expression is also seen in dilated cardiomyopathy, which is far more common than ACM. Conduction abnormalities are likewise a feature of ZO-1 cardiac-specific knockout (ZO-1cKO) mice. However, the role of ZO-1 in sinoatrial node (SAN) automaticity has not been studied. OBJECTIVETo investigate the role of ZO-1 in SAN automaticity and elucidate the mechanisms by which ZO-1 deficiency leads to SAN dysfunction. METHODSZO-1 cardiac-specific knockout (ZO-1cKO) mice were generated by crossing ZO-1 floxed mice with MHC-nuclear Cre mice. SAN/atrial tissue and isolated SAN cells were examined using optical mapping, single-cell patch clamp, and quantitative PCR techniques to assess functional alterations caused by ZO-1 loss. RESULTSZO-1cKO mice exhibited enlarged atria and SAN area compared to control mice, with normal left ventricular function. Electrocardiograms showed sinus bradycardia, sinus pauses and atrioventricular block. Optical mapping revealed a caudal shift in the SAN leading region and reduced intra-atrial conduction velocity in ZO-1cKO mice. Patch-clamp recordings from isolated SAN cells showed reduced spontaneous action potential frequency and diastolic depolarization rate, while voltage-clamp revealed a marked reduction in pacemaker current (If). CONCLUSIONZO-1 expression is essential for SAN automaticity. Its loss impairs SAN impulse generation by reducing pacemaker current and hampering atrial conduction, leading to bradyarrhythmia, conduction delay and block. These findings help explain impulse generation and conduction abnormalities in ACM and other cardiomyopathies.

physiology↗

bioRxiv2026

Human white matter tracts aligned with canonical functional brain networks

White matter tracts form the structural backbone of large-scale brain networks, yet their relation to functional organization remains poorly defined. Although major pathways are well characterized anatomically, their relationship to distributed cognitive systems has not been systematically established. Here, we constructed a population-level white matter tract termination atlas and projected tract endpoints onto the cortical surface to characterize their spatial organization. We integrated this atlas with large-scale meta-analytic decoding to derive functional profiles for individual tracts. Functional decoding revealed that white matter tracts exhibited distinct and biologically interpretable cognitive signatures. Hierarchical clustering of these profiles further showed that tracts organize into coherent ensembles defined by shared functional associations. These tract ensembles recapitulated canonical intrinsic brain networks across multiple cortical atlases, including a notable ensemble that demonstrated alignment with the default mode, salience, and frontoparietal control networks, corresponding to the core architecture of the triple-network model of cognitive control. This finding identified a candidate structural backbone linking distributed functional systems implicated across neuropsychiatric conditions. Together, these results demonstrate that white matter architecture is organized according to large-scale functional principles and establish a tract-to-network framework for linking structural connectivity to cognition.

neuroscience↗

bioRxiv2026

Whole-brain protein profiling using organ-scale multiplexed immunolabeling and image co-registration

Proteins are major drivers of biological functions. Single-cell, organ-scale multiplexed protein imaging can reveal high-dimensional molecular and structural features of individual cells and their interactions, enabling an in-depth understanding of complex biological systems. However, achieving such imaging has remained elusive due to hurdles in multiplexed immunolabeling (mIF) of intact organs and integrative image analysis. Here, we present 3D CYCLIC, an organ-scale multiplexed immunolabeling technique, and TACTIC, a single-cell-level, organ-scale image co-registration algorithm. 3D CYCLIC combines ultrafast, versatile 3D immunolabeling with a cleavable crosslinker that preserves signals by protecting bound antibodies during optical clearing while enabling their detachment for subsequent rounds of immunolabeling. TACTIC uses deep warping networks coupled with a propagation-based cell-pair search to co-register individual cells across whole-brain images acquired from multiple rounds of 3D CYCLIC labeling. 3D CYCLIC enabled 6-plex protein profiling of a mouse brain hemisphere, with images that can be combined with TACTIC for integrative analysis. 3D CYCLIC and TACTIC are versatile techniques and will therefore enable a holistic, unbiased understanding of diverse organs.

neuroscience↗

bioRxiv2026

Identification of functional non-coding variants affecting Alzheimer's disease risk by Massively Parallel Reporter Assay

The majority of genetic risk variants for late-onset Alzheimers disease (LOAD) reside within non-coding genomic regions, suggesting they exert pathogenic effects by disrupting transcriptional regulatory programs. To systematically identify functional variants, we performed an unbiased, high-throughput Massively Parallel Reporter Assay (MPRA) in the THP-1 human monocytic cell line, screening 2,231 SNPs across 24 LOAD GWAS loci. We identified 62 variants exhibiting significant allele-specific transcriptional regulatory output, including rs636317 in the MS4A locus. The risk allele of rs636317 disrupts a CTCF binding motif, altering fine-scale chromatin looping. To validate these findings, we employed CRISPR/Cas9 to generate an allele-specific deletion of the rs636317 T allele in H9 human embryonic stem cells. Monocytes differentiated from these edited cells displayed increased expression of MS4A4E, MS4A6A, and TREM2, along with highly elevated levels of soluble TREM2 (sTREM2). These results provide a mechanistic link between a non-coding genetic variant, transcriptional regulation of the MS4A family, and TREM2-mediated immune responses in Alzheimers disease pathogenesis.

genetics↗

bioRxiv2026

Arrestin-3 promotes locomotor sensitization to psychostimulants via JNK signaling in nucleus accumbens

Arrestins play key role in desensitization of G protein-coupled receptors. Direct signaling role of arrestins has also been documented. Two ubiquitously expressed arrestin isoforms, arrestin-2 and -3 (Arr3), perform similarly in receptor desensitization and share many signaling functions, enabling them to substitute for one another. However, certain signaling roles are specific to each isoform. Mice lacking Arr3 (A3KO) show blunted acute responsiveness to the locomotor stimulatory effect of amphetamine (AMPH). Here we demonstrate that AMPH- and cocaine-induced locomotion of A3KO mice is significantly reduced. This loss-of-function phenotype suggests that Arr3-mediated signaling contributes to the effect. Virus-driven expression of Arr3 in caudate-putamen of A3KO and wild type mice suppressed AMPH-induced locomotion. In contrast, restoration of Arr3 in nucleus accumbens rescued locomotor response. Thus, in caudate-putamen Arr3 participates in the desensitization of dopamine receptors, whereas Arr3-dependent signaling in nucleus accumbens underlies the molecular mechanism of the locomotor response and sensitization. Using monofunctional Arr3-derived peptides, we showed that in the nucleus accumbens Arr3 promoted drug-induced locomotor responses via facilitation of JNK3 activation.

neuroscience↗

bioRxiv2026

Tuned inhibitory control of neuronal firing thresholds explains predictive sensorimotor behavior

Prior expectations guide sensorimotor behavior when sensory information is uncertain, yet the cellular mechanisms underlying this integration remain elusive. Here, we investigate how priors in sensory motion direction shape neural population dynamics by combining recurrent neural network (RNN) modeling with macaque smooth pursuit behavior and middle temporal area (area MT) electrophysiology. Our RNN model reveals that prior expectations are implemented by elevating firing thresholds in neurons tuned away from the expected direction. This selective inhibition sharpens population tuning and reduces behavioral variability under weak sensory information conditions. We validated this prediction in vivo: delta-band local field potentials in area MT, where the phase reflects neural excitability states, exhibited direction-specific phase shifts that scaled with the deviation from the expected direction. These findings demonstrate that prior expectations enhance behavioral reliability through tuned inhibitory control of neuronal excitability, providing a mechanistic link between Bayesian inference and cortical circuit dynamics.

neuroscience↗

bioRxiv2026

Amplification-Free Detection of Antibiotic Resistance in Enterococcus faecium using PNA-FISH

Vancomycin-resistant Enterococcus faecium (VREfm) is a major nosocomial pathogen, with antibiotic resistance mediated by the vanA and vanB operons. Rapid and accurate detection of antibiotic resistance is critical for the timely treatment of bacteremia and sepsis. Although imaging-based approaches using fluorescence in situ hybridization (FISH) provide a potential diagnostic solution, detecting mRNAs of antibiotic resistance genes (ARGs) in individual cells remains particularly challenging due to their low copy number and transient expression. Here, we present a peptide nucleic acid (PNA)-FISH method for direct detection of vanA- and vanB-associated resistance in individual VREfm cells. A universal probe targeting the conserved region across vancomycin resistance genes and a set of probes exclusively targeting the vanB gene were designed. The universal probe showed increased fluorescence in the vanA-genotype strain upon vancomycin or teicoplanin treatment, and in the vanB-genotype strain upon vancomycin treatment. In contrast, vanB-specific probes showed increased fluorescence exclusively from the vanB-genotype strain upon vancomycin treatment, confirming their specificity to the vanB gene. Efficient cellular penetration and strong hybridization of PNA probes enabled efficient and accurate detection of antibiotic-resistant bacterial cells, even under a wide-field fluorescence microscope. No detectable signals above background were observed in other major bacterial species associated with bacteremia and sepsis. These findings demonstrate robust detection of antibiotic-resistant cells in mixed microbial populations. When integrated with microbe-capturing techniques, this method may support culture-free detection of antibiotic resistance without nucleic acid amplification or sequencing, with the potential to reduce diagnostic turnaround time.

microbiology↗

bioRxiv2026

A SRC-Annexin A2 axis that couples membrane repair to microRNA export during radiation stress in glioblastoma

Repair of ionizing-radiation (IR)-induced membrane damage is essential for cell survival, and Annexin A2 (ANXA2) mediates this repair by promoting shedding of microvesicles containing both damaged lipid and ANXA2. Here, we show that coupling this process to microRNA export contributes to radiation resistance of glioblastoma, the most common adult brain cancer. Structural modeling and mutational analysis identify that ANXA2 tyrosine 23 (Y23) phosphorylation, required for its IR-induced localization to damaged membranes, is also essential for binding miR-603, a critical regulator of glioblastoma cell state. This binding is required for IR-induced miR-603 export, indicating that the repair program recruiting ANXA2 also positions it to load miR-603 into microvesicles. Mutations that abolish ROS-induced sulfenylation and activation of SRC (SRC-C185A/C245A) suppressed ANXA2-Y23 phosphorylation, microvesicle shedding, and miR-603 export. The epistatic interaction between ANXA2-Y23A, SRC-C185A/C245A, and miR-603 delineates a therapeutically targetable radiation-response network that couples membrane repair with miRNA export.

cancer biology↗

bioRxiv2026

Coordinated Temporal Dynamics of Glucocorticoid Receptor Binding and Chromatin Landscape Drive Transcriptional Regulation

Glucocorticoid receptor (GR) signaling elicits diverse transcriptional responses through dynamic and context-dependent interactions with chromatin. Here, we define a temporally resolved and mechanistically integrated framework for GR-mediated gene regulation. Time-resolved analyses identify three conserved classes of GR chromatin binding (sustained, transient, and late), distinguished by differences in motif strength, chromatin accessibility, and cofactors engagement. Early GR binding preferentially occurs at high-affinity glucocorticoid response elements (GREs) within pre-accessible regulatory regions, whereas late binding is associated with weaker motifs and requires chromatin remodeling activity. Enhancer activation, marked by H3K27ac deposition, closely tracks GR occupancy, supporting a model in which GR recruits acetyltransferase activity to drive coordinated enhancer activation. Concurrently, GR-centered interaction networks are dynamically reconfigured, and motif enrichment analyses identify distinct transcription factor signatures across binding classes, including AP-1/JUNB at transient sites and CEBP family members at late-binding regions. Integration of chromatin binding, chromatin interaction, and transcriptomic datasets reveals that temporal and combinatorial GR occupancy is functionally linked to gene expression programs. Distinct GR binding clusters are nonrandomly associated with specific transcriptional trajectories, including sustained, transient, and late gene induction. Moreover, combinatorial occupancy across multiple regulatory elements correlates quantitatively with transcriptional output, indicating that GR functions not as a simple binary regulator, but as an integrator of multilayered regulatory inputs. These findings support a unified model in which temporal binding dynamics, chromatin state, and combinatorial enhancer activity collectively encode transcriptional specificity, providing a general framework for stimulus-responsive nuclear receptor signaling.

molecular biology↗

bioRxiv2026

Orai1-mediated Ca2+ Entry Regulates Lipolysis and Mitochondrial Activation in Brown Adipose Thermogenesis

Cold-induced thermogenesis in brown adipose tissue (BAT) is essential for maintaining energy homeostasis, yet the Ca2+-dependent mechanisms underlying this process remain incompletely understood. Here, we identify Orai1, a component of the store-operated Ca2+ entry pathway, as a regulator of thermogenic activation in BAT. Using a brown adipocyte-specific Orai1 knockout mouse model, we demonstrate that cold exposure is associated with Orai1-dependent Ca2+ influx through a non-canonical mechanism. Orai1 deficiency leads to impaired cAMP-PKA signaling, reduces the expression of lipolytic enzymes and thermogenic genes, and diminished mitochondrial Ca2+ uptake and uncoupling. These defects culminate in cold intolerance, lipid accumulation, and decreased energy expenditure. Mechanistically, Orai1 facilitates Ca2+-dependent activation of adenylyl cyclase 3, linking membrane Ca2+ entry to cAMP production, and promotes mitochondrial remodeling and oxidative metabolism. These findings support a key role for Orai1 in coordinating Ca2+ entry to lipolytic and mitochondrial pathways in brown adipocytes and highlight its potential therapeutic target in metabolic diseases characterized by impaired energy metabolism. HIGHLIGHTSO_LIOrai1 mediates Ca2+ influx in brown adipocytes through a non-canonical, partially STIM1-independent mechanism. C_LIO_LIOrai1-mediated Ca2+ influx promotes both cAMP-PKA-driven lipolysis and mitochondrial oxidative activation. C_LIO_LIOrai1-dependent Ca2+ entry promotes cAMP-PKA signaling and lipolytic activation I nbrown adipocytes. C_LIO_LIOrai1 coordinates mitochondrial Ca2+ uptake to support thermogenic function in brown adipocytes. C_LI

physiology↗

bioRxiv2026

Comparative Analysis of Relative Ligand Binding Free Energy Simulation Methods: Amber-TI, GROMACS-NETI, OpenMM-FEP, and BLaDE-MSLD

Structure-based drug design has become increasingly important in the pharmaceutical industry for accelerating the discovery of effective drug candidates. In particular, ligand binding free energy serves as a critical metric for predicting drug efficacy during the key stages of hit discovery and lead optimization. Continuous progresses have been made in the prediction of ligand binding free energies, but direct comparisons of different methods using the same force field remain challenging due to their unique implementations into different simulation engines. In this study, we present a direct comparison of four popular methodologies (Amber-TI, GROMACS-NETI, OpenMM-FEP, and BLaDE-MSLD) for calculating relative binding free energies ({Delta}{Delta}Gbind) with the same Amber protein and ligand force fields using MolCube Alchemical Free Energy Simulator (MolCube-AFES), which provides an input generation workflow to support {Delta}{Delta}Gbind calculations of all four methods. We used 80 alchemical transformations (among the JACS benchmark set by Wang et al.) and two additional applications to compare the predicted {Delta}{Delta}Gbind from the four methods against experimental measurements. All four methods reproduced experimentally observed trends with most transformations within {+/-}2 kcal/mol from experiments and show broadly comparable accuracy with no statistically significant performance differences across the benchmark dataset. These results demonstrate that MolCube-AFES enables controlled, cross platform benchmarking and show that all four different alchemical free energy methods deliver statistically equivalent accuracy, with method selection guided by workflow requirements such as throughput, portability, and perturbation network design rather than expected differences in performances.

biophysics↗

bioRxiv2026

Food preference is associated with distinct large-scale cortical functional connectivity patterns during food-image observation

Food preference influences behavior toward food-related stimuli, yet the large-scale neural mechanisms underlying this process remain unclear. This study investigated whether preferred and nonpreferred food cues are associated with distinct patterns of cortical functional connectivity during the observation of food images. Data from 25 of the 40 recruited healthy adults were included in the final analysis after excluding individuals with highly unbalanced response tendencies. Participants viewed 150 food images and rated each image on a four-point preference scale. Trials were classified as favorite food (FF) or disliked food (DF). High-density electroencephalography (EEG) was recorded during the task, and source-level ROI-to-ROI functional connectivity was analyzed using amplitude envelope correlation in the alpha (8-13 Hz) and beta (13-25 Hz) frequency bands over the 1000-ms period after food-picture onset. Response time did not differ significantly between FF and DF trials. However, distinct functional connectivity patterns were observed between conditions in both frequency bands. In the alpha band, FF trials involved a network including the cuneus, parietal regions, cingulate regions, and lateral occipital cortex, whereas DF trials involved the isthmus cingulate, caudal middle frontal gyrus, inferior temporal cortex, superior parietal lobule, and lateral occipital cortex. In the beta band, FF trials involved the isthmus cingulate, precuneus, parietal regions, and pericalcarine cortex, whereas DF trials additionally involved frontal regions, including the superior frontal gyrus and pars triangularis. These findings indicate that food preference is associated with distinct large-scale cortical functional connectivity patterns during food image observation, suggesting differential neural processing of preferred and nonpreferred food cues.

neuroscience↗

bioRxiv2026

A High-Throughput Platform for Rapidly Adapting DNA Aptamers to SARS-CoV-2 Evolution

Rapid pathogen evolution threatens public health by eroding the effectiveness of vaccines, therapeutics, and diagnostic tools. Although spike protein targeting monoclonal antibodies (mAbs) were developed within 10-12 months of the initial outbreak to serve as key theranostic agents, their redesign has struggled to keep pace with viral evolution, rendering many neutralizing antibodies ineffective. Here we demonstrate a novel platform that combines a random-rational hybrid library diversification with high-throughput MiSeq screening to rapidly reprogram aptamers against emerging SARS-CoV-2 spike variants. Interactions between 3 different spike proteins and 11,806 unique aptamer variant designs were profiled within a few days. Starting from a 40-nt aptamer originally selected against wild-type (WT) spike protein, our screen identified a Delta-binding mutant with a 4-fold affinity improvement and an Omicron-binding mutant that converted undetectable binding into nanomolar affinity. We also identified a WT-selective mutant with substantially reduced affinity for Delta, as well as previously unrecognized bases that critically contribute to spike recognition. Integrating high-throughput binding data with molecular dynamics simulations further revealed sequence-dependent structural features underlying variant-specific aptamer-spike interactions. Finally, we developed a sensor based on the identified WT-selective aptamer mutant, enabling highly specific detection of the WT spike protein with robust performance. Together, this work establishes a rapid and adaptable aptamer engineering platform for rapid adaptation of aptamers to evolving pathogens in future pandemics.

bioengineering↗