Critical Motif Research Articles

Robust Sequence Design Space for the Isothermal Exponential Amplification of Short Oligonucleotides.

Advances in isothermal amplification techniques have accelerated development in biosensing applications and the design of complex molecular devices. The exponential amplification reaction technique, or EXPAR, is uniquely positioned to process molecular information from short oligonucleotide strands (≈10nucleotides length) typically encountered in molecular computing or microRNA detection. Despite its conceptual simplicity (requiring only a template strand and two enzymes), the issue of nonspecific background amplification has hindered broader adoption. In this work, a new system configuration is established at 37 °C to achieve significantly improved performance. Critical sequence motifs responsible for the excellent signal-to-background profile are identified and generalized as a universal adapter design framework. Orthogonal template sequences generated from the framework are implemented for a triplex reaction and successfully evaluated mixtures of multiple-target inputs in a single-step, one-pot format without the need for exogenous agents.

COMPUTATIONAL ANALYSIS OF PHOSPHORYLATED GSTP1: INSIGHTS INTO ALTERED ACTIVITY UPON BINDING WITH GLUTATHIONE AND ETHACRYNIC ACID

Glutathione S-transferase pi 1 (GSTP1) is an essential detoxifying enzyme that plays a critical role in protecting cells from oxidative stress and carcinogenesis. Employing a molecular dynamics simulations approach, we delineate the specific phosphorylation sites on GSTP1 except the WT. Our study unveils critical residues and structural motifs susceptible to phosphorylation-induced destabilization, offering novel insights into the regulatory mechanisms underlying GSTP1 function and potential avenues for therapeutic intervention in diseases associated with dysregulated GSTP1 activity.

Toll-like receptor 21 in Labeo rohita recognizes double-stranded RNA and lipopolysaccharides by engaging the critical motifs in the LRR domain and gets activated against bacterial assaults

TLR (Toll-like receptor)-21 is a non-mammalian TLR and exhibits a unique function within the innate immune systems of fishes, birds, and amphibians. Despite its important role as PRR (pattern recognition receptor), research on TLR21 in many fish species, as well as in rohu (Labeo rohita), remains relatively limited. This article describes the molecular cloning of LrTLR21 (TLR21 in L. rohita), 3D (3-dimensional) modelling of its LRR (leucine-rich repeat)-regions, prediction of LRR18 to LRR20 as the LPS (lipopolysaccharide)-binding site, and LRR1 to LRR5 and LRR21 to LRR23 as the poly I:C (polyinosinic-polycytidylic acid) binding sites. It also describes the response of LrTLR21 in response to Aeromonas hydrophila and Edwardsiella tarda infections and PAMPs (pathogen-associated molecular patterns) (LPS and poly I:C)-stimulations. The ORF (open reading frame) of the LrTLR21 comprises 2955 nucleotides, encoding 984 aa (amino acid) residues with molecular weight and isoelectric point of 113.791 kDa and ∼8.79, respectively. Domain analysis of the deduced LrTLR21 displayed the existence of a signal peptide (residues 1–24), 26 LRR regions (residues 61–685), a TM (transmembrane) domain (residues 736–758), and a TIR (Toll/interleukin-1 receptor) domain (residues 790–937). The 3D model of LrTLR21-LRR regions has parallel β-sheets and few α-helices. Phylogenetically, LrTLR21 is closely related to the Onychostoma macrolepis and Carassius gibelio TLR21, and during ontogenesis, it is expressed in most of the developmental stages. In rohu fingerlings, it is consistently expressed in all examined tissues viz., skin, liver, heart, blood, eye, muscle, kidney, intestine, brain, gill, and spleen. Upon exposure to E. tarda and A. hydrophila infections, as well as LPS and poly I:C stimulations, the expression of LrTLR21 is significantly (p < 0.05) increased in the blood, kidney, liver, and gill. In the RBCs (red blood cells), PBLs (peripheral blood leukocytes), and kidney macrophages, LrTLR21 is also significantly induced following in-vivo and in-vitro LPS and poly I: C-stimulations. These findings on LrTLR21 are the first ones showing its structural insights and PAMPs binding motifs and its key role in recognizing pathogens and their PAMPs in RBCs, PBLs, and macrophages.

Jesus vs Seth-Typhon: An Audience-Critical Approach to Mark 4:35–5:20

Abstract Scholars have approached the Gerasene Demoniac pericope (Mark 5:1–20; = GDP) in light of various traditions, such as the Homeric parallels, the Watcher (i.e. Enochic) story, the scapegoat motif, and imperial-critical and gendered perspectives. Without denying the validity of these attempts, I suggest that there is still an important—but largely ignored—set of traditions that deserves our attention: the Seth-Typhon traditions. Based on an audience-critical perspective, this article suggests that several elements in GDP could allude to the Seth-Typhon traditions in the minds of the Greco-Roman audience of Mark. The strength of this method is twofold. First, it accounts for the close relationship between GDP and its preceding pericope (i.e. the Storm-Stilling pericope; Mark 4:35–41). Second, it coherently explains multiple critical motifs—the crossing of the sea, the sleeping Jesus, the attempt to drown Jesus, the sea-storm, the foreign area, the legion of demons, and the death of the pigs—featured in Mark 4:35–5:20. According to this approach, Jesus in Mark 4:35–5:20 can be read as a divine warrior who outshines his pagan counterparts (i.e. Zeus and Osiris) as he battles against the cosmic forces of evil and eventually conquers them.

Inducible gene expression of IκB-kinase ε is dependent on nuclear factor-κB in human pulmonary epithelial cells.

While IκB-kinase-ε (IKKε) induces immunomodulatory genes following viral stimuli, its up-regulation by inflammatory cytokines remains under-explored. Since airway epithelial cells respond to airborne insults and potentiate inflammation, IKKε expression was characterized in pulmonary epithelial cell lines (A549, BEAS-2B) and primary human bronchial epithelial cells grown as submersion or differentiated air-liquid interface cultures. IKKε expression was up-regulated by the pro-inflammatory cytokines, interleukin-1β (IL-1β) and tumour necrosis factor-α (TNFα). Thus, mechanistic interrogations in A549 cells were used to demonstrate the NF-κB dependence of cytokine-induced IKKε. Furthermore, chromatin immunoprecipitation in A549 and BEAS-2B cells revealed robust recruitment of the NF-κB subunit, p65, to one 5' and two intronic regions within the IKKε locus (IKBKE). In addition, IL-1β and TNFα induced strong RNA polymerase 2 recruitment to the 5' region, the first intron, and the transcription start site. Stable transfection of the p65-binding regions into A549 cells revealed IL-1β- and TNFα-inducible reporter activity that required NF-κB, but was not repressed by glucocorticoid. While critical NF-κB motifs were identified in the 5' and downstream intronic regions, the first intronic region did not contain functional NF-κB motifs. Thus, IL-1β- and TNFα-induced IKKε expression involves three NF-κB-binding regions, containing multiple functional NF-κB motifs, and potentially other mechanisms of p65 binding through non-classical NF-κB binding motifs. By enhancing IKKε expression, IL-1β may prime, or potentiate, responses to alternative stimuli, as modelled by IKKε phosphorylation induced by phorbol 12-myristate 13-acetate. However, since IKKε expression was only partially repressed by glucocorticoid, IKKε-dependent responses could contribute to glucocorticoid-resistant disease.

Regioselective Annulation of 6-Carboxy-Substituted Pyrones as a Two-Carbon Unit in Formal [4 + 2] Cycloaddition Reactions.

Heterocycles serve as a critical motif in chemistry, but despite being present in more than 85% of pharmaceuticals, there are limited methods for their construction. Here, we describe the incorporation of intact pyrone (2H-pyran-2-one) into larger ring systems via annulation. In a formal [4 + 2] cycloaddition, the pyrone regioselectively accepts a benzylic anion as a nucleophile in a conjugate addition fashion, with the subsequent pyrone-derived enolate attaching to a pendant ester on the initial nucleophile. Subsequent base-driven enolate formation and elimination establish aromaticity of the newly formed ring. After optimization of this process using an NMR-based assessment to overcome solubility and separation challenges, the reaction was successfully applied to a library of 6-ester and -amide-substituted pyrones and using a phenyl ester and other substituted sulfoxides. This technology enables the incorporation of intact pyrone rings into more complex systems, such as for the total synthesis of the natural product thermorubin.

Gap-junction-mediated bioelectric signaling required for slow muscle development and function in zebrafish

Bioelectric signaling, intercellular communication facilitated by membrane potential and electrochemical coupling, is emerging as a key regulator of animal development. Gap junction (GJ) channels can mediate bioelectric signaling by creating a fast, direct pathway between cells for the movement of ions and other small molecules. In vertebrates, GJ channels are formed by a highly conserved transmembrane protein family called the connexins. The connexin gene family is large and complex, creating challenges in identifying specific connexins that create channels within developing and mature tissues. Using the embryonic zebrafish neuromuscular system as a model, we identify a connexin conserved across vertebrate lineages, gjd4, which encodes the Cx46.8 protein, that mediates bioelectric signaling required for slow muscle development and function. Through mutant analysis and invivo imaging, we show that gjd4/Cx46.8 creates GJ channels specifically in developing slow muscle cells. Using genetics, pharmacology, and calcium imaging, we find that spinal-cord-generated neural activity is transmitted to developing slow muscle cells, and synchronized activity spreads via gjd4/Cx46.8 GJ channels. Finally, we show that bioelectrical signal propagation within the developing neuromuscular system is required for appropriate myofiber organization and that disruption leads to defects in behavior. Our work reveals a molecular basis for GJ communication among developing muscle cells and reveals how perturbations to bioelectric signaling in the neuromuscular system may contribute to developmental myopathies. Moreover, this work underscores a critical motif of signal propagation between organ systems and highlights the pivotal role of GJ communication in coordinating bioelectric signaling during development.

CDK2 and CDK4: Cell Cycle Functions Evolve Distinct, Catalysis-Competent Conformations, Offering Drug Targets.

Cyclin-dependent kinases (CDKs), particularly CDK4 and CDK2, are crucial for cell cycle progression from the Gap 1 (G1) to the Synthesis (S) phase by phosphorylating targets such as the Retinoblastoma Protein (Rb). CDK4, paired with cyclin-D, operates in the long G1 phase, while CDK2 with cyclin-E, manages the brief G1-to-S transition, enabling DNA replication. Aberrant CDK signaling leads to uncontrolled cell proliferation, which is a hallmark of cancer. Exactly how they accomplish their catalytic phosphorylation actions with distinct efficiencies poses the fundamental, albeit overlooked question. Here we combined available experimental data and modeling of the active complexes to establish their conformational functional landscapes to explain how the two cyclin/CDK complexes differentially populate their catalytically competent states for cell cycle progression. Our premise is that CDK catalytic efficiencies could be more important for cell cycle progression than the cyclin-CDK biochemical binding specificity and that efficiency is likely the prime determinant of cell cycle progression. We observe that CDK4 is more dynamic than CDK2 in the ATP binding site, the regulatory spine, and the interaction with its cyclin partner. The N-terminus of cyclin-D acts as an allosteric regulator of the activation loop and the ATP-binding site in CDK4. Integrated with a suite of experimental data, we suggest that the CDK4 complex is less capable of remaining in the active catalytically competent conformation, and may have a lower catalytic efficiency than CDK2, befitting their cell cycle time scales, and point to critical residues and motifs that drive their differences. Our mechanistic landscape may apply broadly to kinases, and we propose two drug design strategies: (i) allosteric Inhibition by conformational stabilization for targeting allosteric CDK4 regulation by cyclin-D, and (ii) dynamic entropy-optimized targeting which leverages the dynamic, entropic aspects of CDK4 to optimize drug binding efficacy.

Structural insights into PPP2R5A degradation by HIV-1 Vif.

HIV-1 Vif recruits host cullin-RING-E3 ubiquitin ligase and CBFβ to degrade the cellular APOBEC3 antiviral proteins through diverse interactions. Recent evidence has shown that Vif also degrades the regulatory subunits PPP2R5(A-E) of cellular protein phosphatase 2A to induce G2/M cell cycle arrest. As PPP2R5 proteins bear no functional or structural resemblance to A3s, it is unclear how Vif can recognize different sets of proteins. Here we report the cryogenic-electron microscopy structure of PPP2R5A in complex with HIV-1 Vif-CBFβ-elongin B-elongin C at 3.58 Å resolution. The structure shows PPP2R5A binds across the Vif molecule, with biochemical and cellular studies confirming a distinct Vif-PPP2R5A interface that partially overlaps with those for A3s. Vif also blocks a canonical PPP2R5A substrate-binding site, indicating that it suppresses the phosphatase activities through both degradation-dependent and degradation-independent mechanisms. Our work identifies critical Vif motifs regulating the recognition of diverse A3 and PPP2R5A substrates, whereby disruption of these host-virus protein interactions could serve as potential targets for HIV-1 therapeutics.

High-resolution comparative atomic structures of two Giardiavirus prototypes infecting G. duodenalis parasite.

The Giardia lamblia virus (GLV) is a non-enveloped icosahedral dsRNA and endosymbiont virus that infects the zoonotic protozoan parasite Giardia duodenalis (syn. G. lamblia, G. intestinalis), which is a pathogen of mammals, including humans. Elucidating the transmission mechanism of GLV is crucial for gaining an in-depth understanding of the virulence of the virus in G. duodenalis. GLV belongs to the family Totiviridae, which infects yeast and protozoa intracellularly; however, it also transmits extracellularly, similar to the phylogenetically, distantly related toti-like viruses that infect multicellular hosts. The GLV capsid structure is extensively involved in the longstanding discussion concerning extracellular transmission in Totiviridae and toti-like viruses. Hence, this study constructed the first high-resolution comparative atomic models of two GLV strains, namely GLV-HP and GLV-CAT, which showed different intracellular localization and virulence phenotypes, using cryogenic electron microscopy single-particle analysis. The atomic models of the GLV capsids presented swapped C-terminal extensions, extra surface loops, and a lack of cap-snatching pockets, similar to those of toti-like viruses. However, their open pores and absence of the extra crown protein resemble those of other yeast and protozoan Totiviridae viruses, demonstrating the essential structures for extracellular cell-to-cell transmission. The structural comparison between GLV-HP and GLV-CAT indicates the first evidence of critical structural motifs for the transmission and virulence of GLV in G. duodenalis.

Assembly of SARS-CoV-2 nucleocapsid protein with nucleic acid.

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38±10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral andmutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsidprotein variants in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

Versatile Triblock Peptide Self-Assembly System to Mimic Collagen Structure and Function.

The construction of collagen mimetic peptides has been a hot topic in tissue engineering due to their attractive advantages, such as virus-free nature and low immunogenicity. However, all of the reported self-assembled peptides rely on the inclusion of risky elements of potential safety concerns or lack the capability of incorporating critical functional motifs. A versatile self-assembly design of pure synthetic peptides that can mimic the collagen structure and function remains an insurmountably challenging target. We have herein created a type of triblock peptide consisting of a central triple helical block and N-terminal/C-terminal blocks with oppositely charged amino acids. Favorable electrostatic interactions between the two terminal blocks have been demonstrated to trigger the triblock peptides to form collagen-like nanofibers with a distinct D-banding pattern. A length of 3 or above charged amino acid pairs as well as the maintenance of the triple helical conformation are required for the self-assembly of triblock peptides. Notably, integrin and discoidin domain receptor (DDR) binding sequences GFOGER and GVMGFO have been well demonstrated as vivid examples of convenient incorporation of functional motifs into the triblock peptides without interfering with their self-assembly. These triblock peptides provide a robust and versatile strategy to create next-generation peptide-based biomaterials that can recapitulate the structure and function of collagen, which have promising applications in the fields of tissue engineering and regenerative medicine.

Structure and Substrate Specificity of S-Methyl Thiourocanate Hydratase.

Nicotinamide adenine dinucleotide (NAD+) is a common cofactor in enzyme-catalyzed reactions that involve hydride transfers. In contrast, urocanase and urocanase-like enzymes use NAD+ for covalent electrophilic catalysis. Deciphering avenues by which this unusual catalytic strategy has diversified by evolution may point to approaches for the design of novel enzymes. In this report, we describe the S-methyl thiourocanate hydratase (S-Me-TUC) from Variovorax sp. RA8 as a novel member of this small family of NAD+-dependent hydratases. This enzyme catalyzes the 1,4-addition of water to S-methyl thiourocanate as the second step in the catabolism of S-methyl ergothioneine. The crystal structure of this enzyme in complex with the cofactor and a product analogue identifies critical sequence motifs that explain the narrow and nonoverlapping substrate scopes of S-methyl thiourocanate-, urocanate-, thiourocanate-, and Nτ-methyl urocanate-specific hydratases. The discovery of a S-methyl ergothioneine catabolic pathway also suggests that S-methylation or alkylation may be a significant activity in the biology of ergothioneine.

Employing bimodal representations to predict DNA bendability within a self-supervised pre-trained framework.

The bendability of genomic DNA, which measures the DNA looping rate, is crucial for numerous biological processes of DNA. Recently, an advanced high-throughput technique known as 'loop-seq' has made it possible to measure the inherent cyclizability of DNA fragments. However, quantifying the bendability of large-scale DNA is costly, laborious, and time-consuming. To close the gap between rapidly evolving large language models and expanding genomic sequence information, and to elucidate the DNA bendability's impact on critical regulatory sequence motifs such as super-enhancers in the human genome, we introduce an innovative computational model, named MIXBend, to forecast the DNA bendability utilizing both nucleotide sequences and physicochemical properties. In MIXBend, a pre-trained language model DNABERT and convolutional neural network with attention mechanism are utilized to construct both sequence- and physicochemical-based extractors for the sophisticated refinement of DNA sequence representations. These bimodal DNA representations are then fed to a k-mer sequence-physicochemistry matching module to minimize the semantic gap between each modality. Lastly, a self-attention fusion layer is employed for the prediction of DNA bendability. In conclusion, the experimental results validate MIXBend's superior performance relative to other state-of-the-art methods. Additionally, MIXBend reveals both novel and known motifs from the yeast. Moreover, MIXBend discovers significant bendability fluctuations within super-enhancer regions and transcription factors binding sites in the human genome.

The Nuclear Localization Signal of Porcine Circovirus Type 4 Affects the Subcellular Localization of the Virus Capsid and the Production of Virus-like Particles.

Porcine circovirus 4 (PCV4) is a newly identified virus belonging to PCV of the Circoviridae family, the Circovirus genus. We previously found that PCV4 is pathogenic in vitro, while the virus's replication in cells is still unknown. In this study, we evaluated the N-terminal of the PCV4 capsid (Cap) and identified an NLS at amino acid residues 4-37 of the N-terminus of the PCV4 Cap, 4RSRYSRRRRNRRNQRRRGLWPRASRRRYRWRRKN37. The NLS was further divided into two fragments (NLS-A and NLS-B) based on the predicted structure, including two α-helixes, which were located at 4RSRYSRRRRNRRNQRR19 and 24PRASRRRYRWRRK36, respectively. Further studies showed that the NLS, especially the first α-helixes formed by the NLS-A fragment, determined the nuclear localization of the Cap protein, and the amino acid 4RSRY7 in the NLS of the PCV4 Cap was the critical motif affecting the VLP packaging. These results will provide a theoretical basis for elucidating the infection mechanism of PCV4 and developing subunit vaccines based on VLPs.

Structural basis of the human NAIP/NLRC4 inflammasome assembly and pathogen sensing.

The NLR family caspase activation and recruitment domain-containing 4 (NLRC4) inflammasome is a critical cytosolic innate immune machine formed upon the direct sensing of bacterial infection and in response to cell stress during sterile chronic inflammation. Despite its major role in instigating the subsequent host immune response, a more complete understanding of the molecular events in the formation of the NLRC4 inflammasome in humans is lacking. Here we identify Bacillus thailandensis type III secretion system needle protein (Needle) as a potent trigger of the human NLR family apoptosis inhibitory protein (NAIP)/NLRC4 inflammasome complex formation and determine its structural features by cryogenic electron microscopy. We also provide a detailed understanding of how type III secretion system pathogen components are sensed by human NAIP to form a cascade of NLRC4 protomer through a critical lasso-like motif, a 'lock-key' activation model and large structural rearrangement, ultimately forming the full human NLRC4 inflammasome. These results shed light on key regulatory mechanisms specific to the NLRC4 inflammasome assembly, and the innate immune modalities of pathogen sensing in humans.

A kinetic model reveals the critical gating motifs for donor-substrate loading into Actinobacillus pleuropneumoniae N-glycosyltransferase.

Soluble N-glycosyltransferase from Actinobacillus pleuropneumoniae (ApNGT) catalyzes the glycosylation of asparagine residues, and represents one of the most encouraging biocatalysts for N-glycoprotein production. Since the sugar tolerance of ApNGT is restricted to limited monosaccharides (e.g., Glc, GlcN, Gal, Xyl, and Man), tremendous efforts are devoted to expanding the substrate scope of ApNGT via enzyme engineering. However, rational design of novel NGT variants suffers from an elusive understanding of the substrate-binding process from a dynamic point of view. Here, by employing extensive all-atom molecular dynamics (MD) simulations integrated with a kinetic model, we reveal, at the atomic level, the complete donor-substrate binding process from the bulk solvent to the ApNGT active-site, and the key intermediate states of UDP-Glc during its loading dynamics. We are able to determine the critical transition event that limits the overall binding rate, which guides us to pinpoint the key ApNGT residues dictating the donor-substrate entry. The functional roles of several identified gating residues were evaluated through site-directed mutagenesis and enzymatic assays. Two single-point mutations, N471A and S496A, could profoundly enhance the catalytic activity of ApNGT. Our work provides deep mechanistic insights into the structural dynamics of the donor-substrate loading process for ApNGT, which sets a rational basis for design of novel NGT variants with desired substrate specificity.

GASZ self-interaction clusters mitochondria into the intermitochondrial cement for proper germ cell development.

Mitochondrial features and activities vary in a cell type- and developmental stage-dependent manner to critically impact cell function and lineage development. Particularly in male germ cells, mitochondria are uniquely clustered into intermitochondrial cement (IMC), an electron-dense granule in the cytoplasm to support proper spermatogenesis. But it remains puzzling how mitochondria assemble into such a stable structure as IMC without limiting membrane during development. Here, we showed that GASZ (germ cell-specific, ankyrin repeat, SAM and basic leucine zipper domain containing protein), a mitochondrion-localized germ cell-specific protein, self-interacted with each other to cluster mitochondria and maintain protein stability for IMC assembling. When the self-interaction of GASZ was disrupted by either deleting its critical interaction motif or using a blocking peptide, the IMC structure was destabilized, which in turn led to impaired spermatogenesis. Notably, the blocked spermatogenesis was reversible once GASZ self-interaction was recovered. Our findings thus reveal a critical mechanism by which mitochondrion-based granules are properly assembled to support germ cell development while providing an alternative strategy for developing nonhormonal male contraceptives by targeting IMC protein interactions.

FSO2 Radical-Initiated Photoredox Cyclization of 4-Enoic Acids to Functionalized γ-Lactones

AbstractThe incorporation of sulfonyl fluoride groups into molecules has been proven effective in enhancing their biological activities or introducing new functions. Herein, a transition-metal-free and visible-light-mediated radical tandem cyclization of unsaturated carboxylic acid is reported. This affords a facile access to FSO2-functionalized γ-lactones efficiently, which are critical structural motifs widely present in biologically active molecules.

Selection, activity, and nuclease stabilization of cross-neutralizing DNA aptamers targeting HSV-1 and HSV-2

Cross-neutralizing aptamers targeting both HSV-1 and HSV-2 were developed by selecting against the ectodomains of glycoprotein D (gD) from both viruses in parallel as well as sequentially using the SELEX method. Since gD facilitates viral invasion, sterically blocking the host-receptor interaction prevents infection. Candidate aptamers were screened, and lead aptamers were identified that exhibited exceptional neutralizing activity against both viruses in vitro. The specificity of the aptamers was confirmed by comparing their activity to scrambled versions of themselves. Modifications of the lead compounds were tested to define critical motifs to guide development. Stability of the aptamers was increased using phosphorothioate backbone linkages, and 2’ methoxy substitutions of terminal and key internal bases. Aptamers were applied in a guinea pig vaginal HSV-2 infection model and found to reduce both the viral load of infected animals and the severity of the resulting disease. These results suggest that cross-neutralizing aptamers can be developed into on-demand antiviral interventions effective against both HSV-1 and HSV-2.