RNA-seq pathway mapping coupled with large-scale molecular-dynamics mechanistic

This project aims to decode the downstream consequences of shelterin dysfunction by integrating transcriptome-wide profiling with atomistic simulation to connect phenotype-pathway-mechanism in a single reproducible framework. In the experimental arm, RNA-seq will be used to capture global transcriptional responses following chemical perturbation of shelterin-associated processes, generating a high-resolution map of stress signaling and cell-fate programs triggered by telomere uncapping–like states. Computationally intensive NGS processing will include rigorous quality control, adapter/quality trimming, alignment or pseudo-alignment–based quantification, generation of gene- and transcript-level count matrices, normalization and batch-aware differential expression, and time-/dose-stratified comparisons where relevant. Downstream analysis will extend beyond DEG lists to pathway-centric interpretation using gene set enrichment (GO/KEGG/Reactome), transcription factor and upstream regulator inference, network and module discovery, and signature comparisons against curated DDR/replication-stress/telomere-maintenance gene sets to determine whether ATM/ATR-like programs, inflammatory/SASP-like programs, or checkpoint/cell-cycle remodeling dominate across conditions. In parallel, molecular-dynamics simulations will provide a mechanistic explanation for how small molecules perturb shelterin interfaces by testing the stability and dynamics of protein–ligand and protein–protein contacts in explicit solvent, running replicated trajectories for statistical confidence, and extracting quantitative descriptors such as contact persistence, hydrogen-bond networks, conformational shifts, pocket hydration changes, and allosteric coupling patterns. Where appropriate, binding energetics will be estimated using endpoint free-energy approaches and/or enhanced sampling to compare derivative chemotypes and prioritize interaction determinants. The integration step will link MD-derived interaction fingerprints to RNA-seq pathway outputs (e.g., correlating interface destabilization patterns with specific transcriptional branches like checkpoint enforcement, senescence programs, or replication-associated stress responses), producing a ranked set of testable hypotheses and a coherent mechanism-of-action model. Deliverables will include fully reproducible analysis pipelines (containerized, version-controlled), curated RNA-seq outputs (counts, DE results, enrichment tables, network modules), MD trajectories and mechanistic summaries (interaction maps, stability metrics, energetics), and an integrated report that identifies convergent downstream signatures of shelterin dysfunction and the structural features most predictive of those signatures.