Hughes Jason J, Kesler Benjamin K, Adams John E, Hospelhorn Blythe G, Neuert Gregor
Department of Molecular Physiology and Biophysics, School of Medicine, Vanderbilt University, Nashville, TN 37232, USA.
Vanderbilt Genetics Institute, School of Medicine, Vanderbilt University, Nashville, TN 37232, USA.
bioRxiv. 2025 Aug 19:2025.08.14.670355. doi: 10.1101/2025.08.14.670355.
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.
单分子RNA荧光原位杂交（smRNA-FISH）是一种广泛应用的方法，用于在细胞和组织中以高空间分辨率可视化和定量RNA分子。该技术依赖于与靶RNA杂交的荧光标记寡核苷酸探针。准确的定量取决于高探针特异性，以确保荧光信号反映靶RNA结合而非非靶标相互作用。许多因素，包括基因组序列复杂性、二级探针结构、杂交条件以及不同细胞类型和细胞系之间的基因表达变异性，都会影响smRNA-FISH探针的效能。现有的smRNA-FISH探针设计工具存在局限性，包括启发式方法狭窄、非靶标评估不完整以及依赖“试错法”。为了应对这些挑战，我们开发了TrueProbes，这是一个探针设计软件平台，它将基于全基因组BLAST的结合分析与热力学建模相结合，以生成高特异性探针集。TrueProbes根据预测的结合亲和力、靶标特异性和结构限制对探针进行排名和选择。它还纳入了一个热力学动力学模拟模型，以提供预测性设计指标，并在用户定义的条件下优化探针性能。我们将TrueProbes与几种广泛使用的smRNA-FISH设计工具进行了基准测试，发现它在多个计算指标和实验验证试验中始终优于其他工具。用TrueProbes设计的探针显示出增强的靶标选择性和卓越的实验性能。
