biostudies-literatureRen YFundamental Research Funds for the Central UniversitiesCancer Research UKKey Research and Development Program of Guangdong ProvinceNational Natural Science Foundation of ChinaWenzhou Institute, University of Chinese Academy of SciencesWellcome Trustgkae1218https://www.ebi.ac.uk/biostudies/studies/S-EPMC11797044biostudies-literatureUnknown53(3)X-ray crystallography is a fundamental technique that provides atomic-level insights into RNA structures. However, obtaining crystals of RNA structures diffracting to high resolution is challenging. We introduce a simple strategy to enhance the resolution limit of RNA crystals by the selective substitution of Watson-Crick pairs by GU pairs within RNA sequences. Our approach has successfully yielded high-resolution structures for eight unique RNA crystals. Notably, six instances showed marked resolution enhancement upon GC/AU to GU base pair substitution, with two cases achieving high-resolution structures from initially poor data. In one case, reverting GU to GC base pairs also improved resolution. Our method facilitated the first structural determinations of the Long Interspersed Nuclear Nucleic acids researchA general strategy for engineering GU base pairs to facilitate RNA crystallization.PMC11797044223507101823217119123ptpy41A186042020B12120300042023B12120600132022B0202050003WIUCASQD2024002320016392022A1515010328Peng XWesthof ELilley DMJZhang ZRen YLiao WDeng JLin XHuang LZhou YWang JZhan JfalseA general strategy for engineering GU base pairs to facilitate RNA crystallization.X-ray crystallography is a fundamental technique that provides atomic-level insights into RNA structures. However, obtaining crystals of RNA structures diffracting to high resolution is challenging. We introduce a simple strategy to enhance the resolution limit of RNA crystals by the selective substitution of Watson-Crick pairs by GU pairs within RNA sequences. Our approach has successfully yielded high-resolution structures for eight unique RNA crystals. Notably, six instances showed marked resolution enhancement upon GC/AU to GU base pair substitution, with two cases achieving high-resolution structures from initially poor data. In one case, reverting GU to GC base pairs also improved resolution. Our method facilitated the first structural determinations of the Long Interspersed Nuclear 2025-01-01T00:00:00Z2025 Jan2026-06-02T04:15:39.597Z2025-04-06T10:12:34.869ZS-EPMC117970443972159210.1093/nar/gkae1218