4MRNA is an abbreviation for “Massive Multi-type Model Molecular Replacement for Nucleic Acids”. This is a novel method designed to enhance molecular replacement (MR) for phasing in X-ray crystallography of nucleic acids.

Applying MR, which is widely used in structure determination (phase determination), to nucleic acids presents unique challenges that are not encountered with proteins. To overcome these issues, we developed an innovative strategy termed 4MRNA.

One major difficulty is that nucleic acids can adopt different 3D structures even with the same sequence. As a result, models existing in the database may not be suitable as search models, and sequence-based structure prediction methods such as AlphaFold are also limited in applicability. Moreover, it is empirically known that MR of nucleic acids can fail even when the search model and the target structure differ only slightly. To address these issues, the new strategy 4MRNA includes creating a large number of diverse search models (= massive multi-type models) and applying them to MR.

We found that by varying three out of the twelve parameters that control the three-dimensional structures of nucleic acids according to different patterns, the resulting set of models included ones that closely matched the correct structure, thereby increasing the success rate of MR.

Building on this strategy, we employed 3DNA, which is a software that generates nucleic acid structural models based on parameters, to create a wide variety of models. The processes of parameter adjustment and model creation have been automated using Shell scripts. Subsequently, MR is carried out for each of the many created models. Since this operation must be repeated many times, we also automated this process using Shell scripts.