bioRxiv : the preprint server for biology最新文献

{"title":"A forest is more than its trees: haplotypes and ancestral recombination graphs.","authors":"Halley Fritze, Nathaniel Pope, Jerome Kelleher, Peter Ralph","doi":"10.1101/2024.11.30.626138","DOIUrl":"10.1101/2024.11.30.626138","url":null,"abstract":"<p><p>Foreshadowing haplotype-based methods of the genomics era, it is an old observation that the \"junction\" between two distinct haplotypes produced by recombination is inherited as a Mendelian marker. In a genealogical context, this recombination-mediated information reflects the persistence of ancestral hap-lotypes across local genealogical trees in which they do not represent coalescences. We show how these non-coalescing haplotypes (\"locally-unary nodes\") may be inserted into ancestral recombination graphs (ARGs), a compact but information-rich data structure describing the genealogical relationships among recombinant sequences. The resulting ARGs are smaller, faster to compute with, and the additional ancestral information that is inserted is nearly always correct where the initial ARG is correct. We provide efficient algorithms to infer locally-unary nodes within existing ARGs, and explore some consequences for ARGs inferred from real data. To do this, we introduce new metrics of agreement and disagreement between ARGs that, unlike previous methods, consider ARGs as describing relationships between haplotypes rather than just a collection of trees.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11888177/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143589276","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Emergence of specific binding and catalysis from a designed generalist binding protein.","authors":"Yuda Chen, Sagar Bhattacharya, Lena Bergmann, Galen J Correy, Sophia K Tan, Kaipeng Hou, Justin Biel, Lei Lu, Ian Bakanas, Alexander N Volkov, Ivan V Korendovych, Nicholas F Polizzi, James S Fraser, William F DeGrado","doi":"10.1101/2025.01.30.635804","DOIUrl":"10.1101/2025.01.30.635804","url":null,"abstract":"<p><p>The evolution of binding and catalysis played a central role in the emergence of life. While natural proteins have finely tuned affinities for their primary ligands, they also bind weakly and promiscuously to other molecules, which serve as starting points for stepwise, incremental evolution of entirely new specificities. Thus, modern proteins emerged from the joint exploration of sequence and structural space. The ability of natural proteins to bind small molecule fragments in well-defined geometries has been widely evaluated using methods including crystallographic fragment screening. However, this approach had not been applied to <i>de novo</i> proteins. Here, we apply this method to explore the binding specificity of a <i>de novo</i> small molecule-binding protein ABLE. As in Nature, we found ABLE was capable of forming weak complexes, which were excellent starting points for designing entirely new functions, including a binder of a turn-on fluorophore and a highly efficient Kemp eliminase enzyme ( <i>k</i> _cat / <i>K</i> _M = 2,200,000 M ^-1 s ^-1 ) approaching the diffusion limit. This work illustrates how simultaneous consideration of both sequence and chemical structure diversity can guide the emergence of new function in designed proteins.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11838529/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143461216","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Susceptible host dynamics explain pathogen resilience to perturbations.","authors":"Sang Woo Park, Bjarke Frost Nielsen, Emily Howerton, Bryan T Grenfell, Sarah Cobey","doi":"10.1101/2025.06.13.659551","DOIUrl":"10.1101/2025.06.13.659551","url":null,"abstract":"<p><p>Interventions to slow the spread of SARS-CoV-2 significantly disrupted the transmission of other pathogens. As interventions lifted, whether and when human pathogens would eventually return to their pre-pandemic dynamics remains to be answered. Here, we present a framework for estimating pathogen resilience based on how fast epidemic patterns return to their pre-pandemic dynamics. By analyzing time series data from Hong Kong, Canada, Korea, and the US, we quantify the resilience of common respiratory pathogens and further predict when each pathogen will eventually return to its pre-pandemic dynamics. Our predictions are able to distinguish which pathogens should have returned already, and deviations from these predictions reveal long-term impacts of pandemic perturbations. We find a faster rate of susceptible replenishment underlies pathogen resilience and sensitivity to both large and small perturbations. Overall, our analysis highlights the persistent nature of common respiratory pathogens compared to vaccine-preventable infections, such as measles.</p><p><strong>Significance statement: </strong>COVID-19 interventions slowed the transmission of other respiratory pathogens, raising questions about the mechanisms driving differences in responses to COVID-19 intervention measures. To address this gap, we characterized pathogen resilience to perturbations by quantifying how fast each pathogen returned to its pre-pandemic epidemic cycles. We applied the resulting framework to data from Hong Kong, Canada, Korea, and the US, and showed that common respiratory pathogens are much more resilient than measles, a vaccine-preventable infection. Finally, we showed that the speed of replenishment of the susceptible population-for example, through waning immunity-largely determines a pathogen's resilience to perturbations, including large interventions and small stochastic changes in the dynamics.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12262668/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144644962","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A myosin hypertrophic cardiomyopathy mutation disrupts the super-relaxed state and boosts contractility by enhanced actin attachment.","authors":"Robert C Cail, Bipasha Barua, Faviolla A Báez-Cruz, Donald A Winkelmann, Yale E Goldman, E Michael Ostap","doi":"10.1101/2025.06.02.657466","DOIUrl":"10.1101/2025.06.02.657466","url":null,"abstract":"<p><p>Hypertrophic cardiomyopathy (HCM) is a leading cause of cardiac failure among individuals under 35. Many genetic mutations that cause HCM enhance ventricular systolic function, suggesting that these HCM mutations are hypercontractile. Among the most common causes of HCM are mutations in the gene MYH7, which encodes for β-cardiac myosin, the principal human ventricular myosin. Previous work has demonstrated that, for purified myosins, some MYH7 mutations are gain-of-function while others cause reduced function, so how they lead to enhanced contractility is not clear. Here, we have characterized the mechanics and kinetics of the severe HCM-causing mutation M493I. Motility assays demonstrate a 70% reduction of actin filament gliding velocities on M493I-coated surfaces relative to WT. This mutation slows ADP release from actomyosin·ADP 5-fold without affecting phosphate release or ATP binding. Yet it enhances steady-state ATPase <i>V _max</i> 2-fold. Through single-molecule mechanical studies, we find that M493I myosin has a normal working stroke of 5 nm but a significantly prolonged actin attachment duration. Under isometric feedback, M493I myosins produce high, sustained force, with an actin detachment rate that is less sensitive to force than that of WT myosin. We also report direct measurement of the equilibrium state of the super-relaxed to disordered relaxed (SRX-DRX) regulatory transition and show its disruption in M493I, with a concomitant enhancement to actin attachment kinetics. Together, these data demonstrate that enhanced myosin binding from inhibition of myosin's off state, combined with slow ADP release and enhanced force production, underlie the enhanced function and etiology of this HCM mutation.</p><p><strong>Significance statement: </strong>Hypertrophic cardiomyopathy (HCM) is a leading genetic cause of sudden cardiac death in young individuals. Although often described as a hypercontractile disease, the molecular basis for this remains unclear, especially for mutations with inhibitory effects in various <i>in vitro</i> assays. We show that the severe HCM mutation M493I in β-cardiac myosin slows ADP release yet enhances force output and actin attachment through multiple mechanisms, including disrupted autoinhibition via the super-relaxed state. Our findings unify seemingly contradictory biophysical changes into a coherent mechanistic model and support the hypothesis that increased myosin head availability, rather than enhanced individual kinetics alone, underlies HCM hypercontractility.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12157379/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144277356","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Large-scale single-molecule analysis of tau proteoforms.","authors":"J Joly, V Budamagunta, Z Zhang, B Nortman, M Jouzi, R Bhatnagar, J D Egertson, M E Flaster, R Grothe, S Guha, K Kaneshige, K McVey, N Nelson, R T Perera, S J Tan, T Trinh, D Arnott, J Lipka, N J Pandya, L Rougé, T J Wendorff, D S Kirkpatrick, A Rohou, D C Butler, S Lotz, A Forton, E R Sartori, J E Schwarz, P S Brereton, K Chen, M A Darcy, H R Golnabi, R Hartley, P F Indermuhl, C E Inman, K M Jin, S Katsyuk, R Kota, B Lowry, J C Menger, D A Miller, M R Newman, A Ogunyemi, J K Robinson, N Steiner, J Sun, S Tabakman, L Wang, Z Wang, S K Wilcox, G T Kapp, S Patel, S Temple, T Bertucci, J Blanchard, A Huhmer, S Sankar, K Juneau, P Mallick","doi":"10.1101/2025.06.26.660445","DOIUrl":"10.1101/2025.06.26.660445","url":null,"abstract":"<p><p>Proteins exist as diverse proteoforms resulting from a combination of genetic variation, alternative splicing, and post-translational modifications. Current methods struggle to capture this complexity at the single-molecule level. Here we introduce Iterative Ma pping of P roteoforms (IMaP), a method that enables the massively-parallel interrogation of millions to billions of single-protein molecules through iterative probing with fluorescently labeled antibodies. Using 12 site-specific antibodies, the method is capable of measuring 2 ¹² (4,096) potential proteoform groups. We used IMaP to measure proteoform group profiles of the tau protein, a key player in neurodegenerative diseases, using two pan anti-tau antibodies (Tau-13, Tau-216), three isoform-specific antibodies (Anti-0N, Anti-2N, Anti-4R), and seven phosphosite-specific antibodies (Anti-pT181, Anti-pS202+pT205, Anti-pT205, Anti-pS214, Anti-pT217, Anti-pT231, and Anti-pS396). The method demonstrates high sensitivity (detecting proteoforms at 0.1% abundance), high reproducibility (median CV <5.5%), and broad dynamic range (>3 orders of magnitude), outperforming conventional techniques in resolving closely related proteoform groups. We demonstrated that the method can be used on relevant biological samples by examining various neuronal models (iNeuron cells, organoids, MiBrains, and mouse brains) and human samples. This examination revealed 130 distinct tau proteoform groups with as many as six phosphorylation events. The non-random distribution of these phosphorylation events suggests ordered and site-specific modification processes rather than random, stochastic accumulation. Certain combinations of phosphorylation events were more abundant than others; for example, pT217 preferentially co-occurred with pT181. In validating the applicability of the assay to human disease samples, we noted a specific pattern of multiple phosphorylation events in an advanced Alzheimer's disease patient that suggests a sequential pathway of pathological tau modification. Iterative Mapping of Proteoforms provides insights into proteoform complexity at the single-molecule level, with significant implications for understanding protein regulation in neurodegenerative diseases and beyond.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12262352/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144644695","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Computer prediction and genetic analysis identifies retinoic acid modulation as a driver of conserved longevity pathways in genetically-diverse <i>Caenorhabditis</i> nematodes.","authors":"Stephen A Banse, Christine A Sedore, Anna L Coleman-Hulbert, Erik Johnson, Brian Onken, David Hall, Erik Segerdell, E Grace Jones, Yuhua Song, Hadley Osman, Jian Xue, Elena Battistoni, Suzhen Guo, Anna C Foulger, Madhuri Achanta, Mustafa Sheikh, Theresa Fitzgibbon, John H Willis, Gavin C Woodruff, Monica Driscoll, Gordon J Lithgow, Patrick C Phillips","doi":"10.1101/2024.10.23.619838","DOIUrl":"10.1101/2024.10.23.619838","url":null,"abstract":"<p><p>Aging is a pan-metazoan process with significant consequences for human health and society-discovery of new compounds that ameliorate the negative health impacts of aging promise to be of tremendous benefit across a number of age-based comorbidities. One method to prioritize a testable subset of the nearly infinite universe of potential compounds is to use computational prediction of their likely anti-aging capacity. Here we present a survey of longevity effects for 16 compounds suggested by a previously published computational prediction set, capitalizing upon the comprehensive, multi-species approach utilized by the <i>Caenorhabditis</i> Intervention Testing Program (CITP). While eleven compounds (aldosterone, arecoline, bortezomib, dasatinib, decitabine, dexamethasone, erlotinib, everolimus, gefitinib, temsirolimus, and thalidomide) either had no effect on median lifespan or were toxic, five compounds (all-trans retinoic acid, berberine, fisetin, propranolol, and ritonavir) extended lifespan in <i>Caenorhabditis elegans</i> . These computer predictions yield a remarkable positive hit rate of 30%. Deeper genetic characterization of the longevity effects of one of the most efficacious compounds, the endogenous signaling ligand all-trans retinoic acid (atRA, designated tretinoin in medical products), which is widely prescribed for treatment of acne, skin photoaging and acute promyelocytic leukemia, demonstrated a requirement for the regulatory kinases AKT-1 and AKT-2. While the canonical Akt-target FOXO/DAF-16 was largely dispensable, other conserved Akt-targets (Nrf2/SKN-1 and HSF1/HSF-1), as well as the conserved catalytic subunit of AMPK AAK-2, were all necessary for longevity extension by atRA. Evolutionary conservation of retinoic acid as a signaling ligand and the structure of the downstream effector network of retinoic acid combine to suggest that the all-trans retinoic acid pathway is an ancient metabolic regulatory system that can modulate lifespan. Our results highlight the potential of combining computational prediction of longevity interventions with the power of nematode functional genetics and underscore that the manipulation of a conserved metabolic regulatory circuit by co-opting endogenous signaling molecules is a powerful approach for discovering aging interventions.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12258912/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144639305","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Scheduled feeding improves behavioral outcomes and reduces inflammation in a mouse model of Fragile X syndrome.","authors":"Huei Bin Wang, Natalie E Smale, Sarah H Brown, Sophia Anne Marie B Villanueva, David Zhou, Aly Mulji, Deap S Bhandal, Kyle Nguyen-Ngo, John R Harvey, Cristina A Ghiani, Christopher S Colwell","doi":"10.1101/2024.09.16.613343","DOIUrl":"10.1101/2024.09.16.613343","url":null,"abstract":"<p><p>Fragile X syndrome (FXS), a leading inherited cause of intellectual disability and autism, is frequently accompanied by sleep and circadian rhythm disturbances. In this study, we comprehensively characterized these disruptions and evaluated the therapeutic potential of a circadian-based intervention in the fragile X mental retardation 1 (FMR1) knockout (KO) mouse. The <i>Fmr1</i> KO mice exhibited fragmented sleep, impaired locomotor rhythmicity, and attenuated behavioral responses to light, linked to an abnormal retinal innervation and reduction of light-evoked neuronal activation in the suprachiasmatic nucleus. Behavioral testing revealed significant deficits in social memory and increased repetitive behaviors in the mutants, which correlated with sleep fragmentation. Remarkably, a scheduled feeding paradigm (6-hour feeding/18-hour fasting) significantly enhanced circadian rhythmicity, consolidated sleep, and improved social deficits and repetitive behaviors in the <i>Fmr1</i> KO mice. This intervention also normalized the elevated levels of some pro-inflammatory cytokines, including IL-12 and IFN-γ, in the mutants' blood, suggesting that its benefits extend to inflammatory pathways. These findings highlight the interplay between circadian disruption, behavior, and an inflammatory response in FXS, and provide compelling evidence that time-restricted feeding may serve as a promising non-pharmacological approach for improving core symptoms in neurodevelopmental disorders.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11429936/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142336137","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cardiac fibroblasts counterbalance cardiomyocytes in <i>LMNA</i> cardiomyopathy pathogenesis.","authors":"Kunal Sikder, Elizabeth Phillips, Nesrine Bouhrira, David Mothy, Nadan Wang, Gisèle Bonne, Kenneth B Margulies, Jason C Choi","doi":"10.1101/2025.06.05.657412","DOIUrl":"10.1101/2025.06.05.657412","url":null,"abstract":"<p><p>Genetic cardiomyopathies arising from mutations in the <i>LMNA</i> gene, encoding nuclear intermediate filaments lamin A/C, display variable age of onset, severity, and fibrosis development. This variability suggests a fundamental element in disease pathogenesis that has yet to be elucidated. Given the central role cardiac fibroblasts play in fibrosis, we explored the relevance of lamin A/C in cardiac fibroblast function, as very little is known in this regard. Using primary cardiac fibroblasts and <i>in vivo</i> mouse models, we show that <i>Lmna</i> mutations impact various aspects of cardiac fibroblast function in response to myocyte damage. We show that both lamin A/C depletion and point-mutant variant expression impair cardiac fibroblast proliferation and contraction whereas other functions such as cell migration appears to be mutation dependent. <i>In vivo</i> depletion of lamin A/C simultaneously in cardiomyocytes and cardiac fibroblasts significantly delayed disease progression, improved cardiac function, and prolonged survival, indicating that lamin A/C mediate an opposing balance between cardiomyocytes and cardiac fibroblasts in driving disease pathogenesis. Our results elucidate previously unexplored roles of lamin A/C in cardiac fibroblasts and suggest that interactions between cardiac fibroblasts and cardiomyocytes are important determinants of the rate of progression and the severity of <i>LMNA</i> cardiomyopathy.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12279081/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144683943","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A mechanism linking dopamine's roles in reinforcement, movement initiation and motivation.","authors":"Allison E Hamilos, Isabella C Wijsman, Qinxin Ding, Pichamon Assawaphadungsit, Zeynep Ozcan, Elias Norri, Kimberly Reinhold, Bernardo L Sabatini, John A Assad","doi":"10.1101/2025.04.04.647288","DOIUrl":"10.1101/2025.04.04.647288","url":null,"abstract":"<p><p>Dopamine neurons (DANs) play seemingly distinct roles in reinforcement, ^1-3 motivation, ^4,5 and movement, ^6,7 and DA-modulating therapies relieve symptoms across a puzzling spectrum of neurologic and psychiatric symptoms. ⁸ Yet, the relationship among these roles is unknown. Here, we show DA's tripartite functions are causally linked by a process in which phasic striatal DA rapidly and persistently recalibrates the propensity to move, a measure of vigor. Using a self-timed movement task, we found that single exposures to reward-related DA transients (both endogenous and exogenously-induced) exerted one-shot updates to movement timing- but in a surprising fashion. Rather than reinforce specific movement times, DA transients quantitatively <i>changed</i> movement timing on the next trial, with larger transients leading to earlier movements (and smaller to later), consistent with a stochastic search process that calibrates the frequency of movement. Both abrupt and gradual changes in external and internal contingencies-such as timing criterion, reward content, and satiety state-caused changes to the amplitude of DA transients that causally altered movement timing. The rapidity and bidirectionality of the one-shot effects are difficult to reconcile with gradual synaptic plasticity, and instead point to more flexible cellular mechanisms, such as DA-dependent modulation of neuronal excitability. Our findings shed light on how natural reinforcement, as well as DA-related disorders such as Parkinson's disease, could affect behavioral vigor.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11996583/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144059547","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Rapid Cell-Free Combinatorial Mutagenesis Workflow Using Small Oligos Suitable for High-Iteration, Active Learning-Guided Protein Engineering.","authors":"Ryan Godin, Sepehr Hejazi, Bret Lange, Basmala Aldamak, Nigel F Reuel","doi":"10.1101/2025.06.18.660386","DOIUrl":"10.1101/2025.06.18.660386","url":null,"abstract":"<p><p>Active learning-guided protein engineering e2iciently navigates the challenging fitness landscape by screening designs iteratively in a model-guided design-build-test-learn cycle. However, while high iterations boost performance, current workflows reliance on tedious and costly cell-based cloning and expression steps limits the iterations they can practically implement. To address this problem, we present a novel combinatorial mutagenesis workflow that uses small (∼20-40 bp) mutagenic annealed-oligo fragments and cell-free expression to rapidly and conveniently screen protein variants in <9 hours. Using bulk-prepared mutagenic oligos eliminates the need for cloning, PCR-based mutagenesis, or ordering costly genes each screening round. Their >80% size reduction from current fragment-based shu2ling strategies also helps avoid including multiple mutations on the same fragment, reducing the number one must order to cover the design space. By screening 3-10 fragment assemblies for two di2erent proteins, we show our approach is a general, scalable, and cost-e2ective platform for high-iteration protein engineering.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12262282/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144644995","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}