Microstate-Guided Design of Minimal Hairpin RNAs

CONTEXT: Short hairpin RNAs with stems of 19 base pairs or less (sshRNAs) can access a high‑potency, Dicer‑independent mode of RNA interference that is poorly captured by conventional shRNA design rules. We introduce Microstate‑Guided Minimal Hairpin Design (MiG‑MHD), a computational framework that links sshRNA architecture to functional microstates governing Ago2 loading, passenger suppression, and innate immune sensing. Across a factorial design space spanning stem length, loop topology, and asymmetric strand designs, MiG‑MHD identifies a narrow but robust regime in which L‑type sshRNAs achieve ultra‑low picomolar IC50 values while remaining minimally immunostimulatory. Potency is best explained by a composite loading‑microstate index that increases with antisense 5′ end openness and controlled asymmetry and decreases with loop‑closure entropy. The framework further predicts that Dicer processing becomes dispensable below approximately 19 base pairs and that designs optimized for Ago2 entry minimize dimerization‑dependence. MiG‑MHD yields quantitative design rules and a candidate panel of minimal hairpins suitable for experimental validation of Dicer‑independent silencing in direct‑delivery contexts. METHODS: A factorial synthetic design library was constructed varying stem length, loop length, orientation, strand asymmetry, GC fraction, and motif composition. MiG‑MHD encodes architectures, computes antisense 5′ end openness and loop‑entropy penalties, and estimates Ago2‑loading propensity and Dicer‑processing probability using logistic and linear models. Potency is modeled as log10(IC50) and candidates are ranked under a risk‑aware objective incorporating an immunostimulation index based on U‑rich motif density and total length. All analyses and visualizations were performed in Python using custom scripts.