Computational evolution of poly(U) polymerase for efficient and controlled RNA oligonucleotide synthesis.

ABSTRACT: Template-independent polymerases such as poly(U) polymerase (PUP) hold promise for enzymatic RNA synthesis but are limited by inefficient incorporation of modified nucleotides. Here, we describe a multi-round, closed-loop workflow integrating Gaussian accelerated molecular dynamics (GaMD), machine learning (ML), and generative artificial intelligence (AI) to engineer PUP variants with enhanced activity and stability. Our engineering strategy commenced with a deep mechanistic analysis of PUP using GaMD simulations. This provided the blueprint for our first key step: engineering PUPdel, a truncated variant that achieved a pivotal breakthrough by incorporating 3'-terminally blocked nucleotides and enabling controlled template-independent synthesis. Subsequently, we screened single-point mutat

SUBMITTER: Yang L 

PROVIDER: S-EPMC12848935 | biostudies-literature | 2026 Jan

REPOSITORIES: biostudies-literature