Thiel, W. H. | Search bioRxiv

bioRxiv2025

ASSET: A Framework for Decoding Aptamer Specificity of an Enriched Library by Next-Generation Sequencing of Experimental Samples

The SELEX process to identify RNA and DNA aptamers relies on sequencing selection rounds to detect highly specific aptamers through patterns of aptamer accumulation or enrichment. However, this approach infers rather than quantify aptamer specificity. Here we present a novel strategy for directly quantifying aptamer specificity within enriched libraries termed Aptamer Specificity Sequencing for Efficient Targeting (ASSET). The ASSET framework takes experimental samples and replicates testing the specificity of an aptamer library and prepares them for next-generation sequencing (NGS) with a known internal reference sequence. This enables robust data normalization, calculation of aptamer specificity scores with statistical significance, and the creation of specificity profiles of individual aptamers across multiple targets and non-targets. By integrating ASSET specificity scores with conventional selection round sequencing data, aptamers can be easily classified as true or false positives and negatives, allowing for easy separation of true positive aptamers. Compared to conventional methods for identifying aptamer candidates, such as measuring abundance or enrichment, ASSET specificity scores show a strong correlation with experimentally measured specificity. This supports ASSET as a more effective metric for selecting lead candidates following SELEX. ASSET is an easily implemented framework that accelerates the identification of highly specific aptamers, thereby expediting aptamer discovery for therapeutic and diagnostic applications.

molecular biology↗

bioRxiv2024

MIRO1 controls energy production and proliferation of smooth muscle cells

BackgroundThe outer mitochondrial Rho GTPase 1, MIRO1, mediates mitochondrial motility within cells, but implications for vascular smooth muscle cell (VSMC) physiology and its roles in vascular diseases, such as neointima formation following vascular injury are widely unknown. MethodsCarotid ligation was performed in an in vivo model of selective Miro1 deletion in smooth muscle cells. VSMC proliferation during the cell cycle and molecular mechanisms of smooth muscle cell proliferation were explored in cultured aortic VSMCs by imaging mitochondrial positioning and cristae structure and assessing the effects on ATP production, metabolic function, and interactions with components of the electron transport chain (ETC). MIRO1 expression was also analyzed in human coronary arteries, and its function was assessed via knockdown in human coronary artery VSMCs. ResultsMIRO1 was highly expressed in VSMCs within human atherosclerotic plaques. MIRO1 facilitated VSMC proliferation and neointima formation by regulating mitochondrial positioning and PDGF-stimulated ATP production and respiration, critical for cell-cycle progression at G1/S. Deletion of Miro1 disrupted mitochondrial cristae structure, diminished ETC complex I activity, and impaired supercomplex formation. Notably, restoring MIRO1 function with expression of wild type MIRO1 recovered proliferation and ATP production and respiration, whereas a mutant lacking EF hands, which are essential for mitochondrial motility, only partially rescued these effects. MIRO1 knockdown in human coronary artery VSMCs confirmed its pivotal role in mitochondrial function and VSMC proliferation. ConclusionsThis study highlights two key mechanisms by which MIRO1 regulates VSMC proliferation. First, it maintains ATP synthesis by preserving mitochondrial cristae integrity. Second, its Ca2+-dependent EF hands enable ATP-dependent mitochondrial positioning. By linking mitochondrial motility and energy production to VSMC physiology, these findings position MIRO1 as a critical regulator of vascular remodeling and a potential target for therapeutic interventions.

cell biology↗