Shin Ando

Structural analysis of nucleic acids lags behind that of proteins, partly because most fundamental structural analysis techniques have been primarily developed for proteins. The molecular replacement (MR) method, commonly used for phase determination in protein crystallography, encounters unique challenges when applied to nucleic acids. Nucleic acids can have different three-dimensional structures even with the same sequence, which often renders database entries or predicted models unsuitable as search models for MR. To overcome the limitation, we developed a novel strategy termed 4MRNA, which stands for Massive Multi-type Model Molecular Replacement for Nucleic Acids. This method introduces a new principle for MR, which is the systematic creation of diverse search models through parameter adjustment. By identifying the parameters that critically influence MR and generating models based on their statistical analysis, 4MRNA can provide search models that closely approximate target structures and thereby improve the success rate of MR. Its effectiveness was validated across comprehensive test cases including canonical duplexes, duplexes with bulges and internal loops, the more complex structure of transfer RNA, and a previously unreported DNA structure. 4MRNA is anticipated to become an indispensable tool for nucleic acid structure determination, profoundly advancing fundamental research and extending its impact to wide-ranging applications including structure-based drug design and nucleic acid nanotechnology.

[Co-first author] Small interfering RNAs (siRNAs) hold significant therapeutic potential but require chemical modifications to achieve efficient delivery and robust RNA interference (RNAi) in target cells. Here, we report a series of newly developed cholesterol-conjugated siRNAs engineered to enable carrier-free cellular uptake and potent gene silencing. Novel 1′-sugar-linked cholesterol derivatives featuring alkyl linkers of 0, 2, 4, or 6 carbons were designed and synthesized. These derivatives (Y1–Y4; Chol-C0, Chol-C2, Chol-C4, and Chol-C6) were conjugated to the 3′ end of the passenger strand to evaluate how linker length affects siRNA duplex stability and RNAi activity. Thermal melting analysis revealed that all cholesterol modifications increased duplex stability, with the Y1 (Chol-C0) conjugate exhibiting the highest Tm, consistent with rigid, linker-free cholesterol positioning at the terminus. X-ray crystallographic studies of Y1-modified oligonucleotides further demonstrated defined cholesterol orientations and extensive sterol–sterol and sterol–nucleobase interactions, providing a structural basis for enhanced duplex stabilization. Functional evaluation in HCT116 colon cancer cells under lipofection-free conditions revealed that cholesterol conjugation markedly improved cellular uptake and gene silencing in a linker-length-dependent manner. The four-carbon linker (Y3: Chol-C4) achieved the most efficient KNTC2 knockdown (∼62% at 200 nM, ∼80% at 500 nM, and ∼93% at 1250 nM), followed closely by the two-carbon linker (Y2: Chol-C2), while shorter (Y1: Chol-C0) or longer (Y4: Chol-C6) linkers exhibited reduced activity. Quantitative IC50 analysis further supported these findings, with Chol-C4 showing the lowest IC50 value (141.5 nM), followed by Chol-C2 (188.4 nM), Chol-C6 (265.4 nM), and Chol-C0 (600.5 nM). This study demonstrates that siRNAs bearing 1′-sugar-tethered cholesterol with optimized linker lengths achieve efficient carrier-free delivery and potent RNAi activity, offering significant promise for advancing siRNA-based therapeutics.

[This paper was selected as the cover illustration.] For the success of structure-based drug design, three-dimensional structures solved by X-ray crystallography at atomic resolution are mandatory. In order to obtain high-quality single crystals with strong diffraction power, crystallization under microgravity conditions has been attempted for proteins. Since nucleic acid duplexes have chemical, structural and crystallographic characteristics that differ from those of globular proteins, such as intermolecular repulsion due to negative charge and molecular and crystallographic anisotropies, it is interesting to investigate whether microgravity crystallization improves the crystal growth of nucleic acids. However, to our knowledge there has been only one report on nucleic acid crystallization in a microgravity environment, and there have been no reports of successful structural analysis. Here, we conducted the crystallization of a DNA/RNA heteroduplex in space. The heteroduplex was successfully crystallized in a microgravity environment, and the size and appearance of the crystals were improved compared with control experiments conducted on Earth. Although the effect of the counter-diffusion method is likely to be more significant than the effect of microgravity in this study, we were able to analyze the structure at a higher resolution (1.4 Å) than our previously reported crystal structure (1.9 Å).