Biology subjects

Kesler, B. K.

Publications and source records attributed to Kesler, B. K..

4 recordsLinked to original sources

bioRxiv2025

TrueProbes: Quantitative Single-Molecule RNA-FISH Probe Design Improves RNA Detection

Single-molecule RNA fluorescence in situ hybridization (smRNA-FISH) is a widely used method for visualizing and quantifying RNA molecules in cells and tissues at high spatial resolution. The technique relies on fluorescently labeled oligonucleotide probes that hybridize to target RNA. Accurate quantification depends on high probe specificity to ensure fluorescent signals reflect target RNA binding rather than off-target interactions. Numerous factors, including genome sequence complexity, secondary probe structure, hybridization conditions, and gene expression variability across cell types and lines, influence smRNA-FISH probe efficacy. Existing smRNA-FISH probe design tools have limitations, including narrow heuristics, incomplete off-target assessment, and reliance on "trial-and-error approaches. To address these challenges, we developed TrueProbes, a probe design software platform that integrates genome-wide BLAST-based binding analysis with thermodynamic modeling to generate high-specificity probe sets. TrueProbes ranks and selects probes based on predicted binding affinity, target specificity, and structural constraints. It also incorporates a thermodynamic-kinetic simulation model to provide predictive design metrics and optimize probe performance under user-defined conditions. We benchmarked TrueProbes against several widely used smRNA-FISH design tools and found that it consistently outperformed alternatives across multiple computational metrics and experimental validation assays. Probes designed with TrueProbes showed enhanced target selectivity and superior experimental performance.

systems biology↗

bioRxiv2025

TrueSpot: A robust automated tool for quantifying signal puncta in fluorescent imaging

Characterizing the movement of biomolecules in single cells quantitatively is essential to understanding fundamental biological mechanisms. RNA fluorescent in situ hybridization (RNA-FISH) is a technique for visualizing RNA in fixed cells using fluorescent probes. Automated processing of the resulting images is essential for large datasets. Here we demonstrate that our RNA-FISH image processing tool, TrueSpot, is useful for automatically detecting the locations of RNA at single molecule resolution. TrueSpot also performs well on images with immunofluorescent (IF) and GFP tagged clustered protein targets. Additionally, we show that our 3D spot detection approach substantially outperforms current 2D spot detection algorithms.

systems biology↗

bioRxiv2025

Transcriptional stochasticity reveals multiple mechanisms of long noncoding RNA regulation at the Xist - Tsix locus

Long noncoding RNAs (LncRNAs) are increasingly recognized as being involved in human physiology and diseases, but there is a lack of mechanistic understanding for the majority of lncRNAs. We comparatively tested proposed mechanisms of antisense lncRNA regulation at the X-chromosome Inactivation (XCI) locus. We find that due to stochasticity in transcription, different mechanisms based on the act of transcription regulate Xist and Tsix at different levels of nascent transcription. At medium levels, RNA polymerases transcribe Xist and Tsix on each strand at the same transcription site and deposit significant amounts of the histone mark H3K36me3, which inhibits Xist. At high levels of nascent transcription, many RNA polymerases transcribe Xist or Tsix resulting in transcriptional interference. Therefore, lncRNA expression variability is not just a quirk of transcription but an important aspect of regulation that allows multiple mechanisms to be employed by the same gene locus within the same cell population.

systems biology↗

bioRxiv2017

Linearly changing stress environment causes cellular growth phenotype

Cells are exposed to changes in extracellular stimulus concentration that vary as a function of rate. However, the effect of stimulation rate on cell behavior and signaling remains poorly understood. Here, we examined how varying the rate of stress application alters budding yeast cell viability and mitogen-activated protein kinase (MAPK) signaling at the single-cell level. We show that cell survival and signaling depend on a rate threshold that operates in conjunction with a concentration threshold to determine the timing of MAPK signaling during rate-varying stimulus treatments. We also discovered that the stimulation rate threshold is sensitive to changes in the expression levels of the Ptp2 phosphatase, but not of another phosphatase that similarly regulates osmostress signaling during switch-like treatments. Our results demonstrate that stimulation rate is a regulated determinant of signaling output and provide a paradigm to guide the dissection of major stimulation rate-dependent mechanisms in other systems.

systems biology↗