近藤 次郎

Gold-mediated base pairing in nucleic acids has remained poorly understood, despite structural analogies with mercury and silver ions known to coordinate selectively to mismatched base pairs. Here, the crystal structures of a CAu(I)C base pair and a CGAu(I)C base triple formed with natural nucleobases are reported. Although solution-phase thermodynamic analysis of Au(I) coordination is technically unfeasible, structural evidence supports its selective insertion into the base mismatches. In contrast, duplexes incorporating 2-thiocytosine form square-planar complexes with Au(III), and melting temperature analysis shows significant thermal stabilization. The distinct coordination geometries of Au(I) and Au(III) arise from differences in oxidation state and preferred coordination numbers, with Au(I) favoring linear two-coordinate structures and Au(III) forming square-planar complexes stabilized by thiocarbonyl donors. These findings establish a structure-guided strategy for oxidation-state-selective metal coordination in nucleic acids, paving the way for the design of metal-responsive DNA architectures with tunable properties.

Tumor stromal remodeling is an obstacle for immune checkpoint inhibitors (ICI). A stroma modifying small interfering RNA (siRNA) to carbohydrate sulfotransferase 15 (CHST15) was recently shown to enhance tumor-infiltrating T cells, yet its impact on antitumor response of ICI remains unexplored. In mouse pancreatic cancer KPC and Pan02 subcutaneous syngeneic tumor models, mice were divided into 4 groups for treatment; (1) control, (2) CHST15 siRNA monotherapy, (3) anti-programmed death receptor 1 (PD-1) monotherapy, and (4) combination therapy with CHST15 siRNA and anti-PD-1 antibody. Mice were sacrificed after 2 week-treatments and anti-tumor effects were evaluated by immunohistochemistry for KPC and flow cytometry for Pan02 model, respectively. In the KPC model, combination treatment with intratumoral CHST15 siRNA (0.9-1.0 mg/kg) and systemic anti-PD-1 antibody (5 mg/kg) synergistically and robustly suppressed tumor growth with a significant increase of tumor-infiltrating CD4+ and CD8+ T cells compared to anti-PD-1 monotherapy. In the Pan02 model, combination treatment with CHST15 siRNA and anti-PD-1 showed anti-tumor effect with significant increases in % necrosis area of the tumor, and tumor-infiltrating T cells compared to the control. Notably, the combination therapy dramatically diminishes Ly6C+Ly6G+ granulocytic myeloid-derived suppressor cells (MDSCs) compared to anti-PD-1 monotherapy. The present study demonstrated the robust synergy between systemic anti-PD-1 antibody and a single stroma modifying agent. Combination usage of intratumoral CHST15 siRNA would provide a novel therapeutic option to trigger the remarkable effect of ICI on this most hard-to-treat solid tumor.

Abstract In this study, we report the synthesis of 2′-formamidonucleoside phosphoramidite derivatives and their incorporation into siRNA strands to reduce seed-based off-target effects of small interfering RNAs (siRNAs). Formamido derivatives of all four nucleosides (A, G, C and U) were synthesized in 5–11 steps from commercial compounds. Introducing these derivatives into double-stranded RNA slightly reduced its thermodynamic stability, but X-ray crystallography and CD spectrum analysis confirmed that the RNA maintained its natural A-form structure. Although the introduction of the 2′-formamidonucleoside derivative at the 2nd position in the guide strand of the siRNA led to a slight decrease in the on-target RNAi activity, the siRNAs with different sequences incorporating 2′-formamidonucleoside with four kinds of nucleobases into any position other than 2nd position in the seed region revealed a significant suppression of off-target activity while maintaining on-target RNAi activity. This indicates that 2′-formamidonucleosides represent a promising approach for mitigating off-target effects in siRNA therapeutics.

The dense stroma is one cause of poor efficacy of T cell-mediated immunotherapy in pancreatic ductal adenocarcinoma (PDAC). Carbohydrate sulfotransferase 15 (CHST15) is a proteoglycan-synthetic enzyme responsible for remodeling tumor stroma. Intra-tumoral injection of CHST15 small interfering RNA (siRNA) has been shown to increase the tumor-infiltrating T cells (TILs) in patients with unresectable PDAC. However, the mechanism underlying the enhanced accumulation of TILs is not fully explored. Here, we demonstrate that intra-tumoral injection of CHST15 siRNA locally and remotely diminishes myeloid-derived suppressor cells (MDSCs) and enhances TILs in mice. CHST15 was expressed by tumor cells and MDSCs in both tumor and tumor-draining lymph nodes (TDLNs), and CHST15 siRNA repressed stromal density, neutrophil extracellular traps, and Ly6C/G+ MDSCs in vivo. Remarkably, tumor growth inhibition was only observed in the immunocompetent KPC model, which is associated with enhanced TILs. In vitro, CHST15 siRNA significantly downregulated the levels of CHST15 and indoleamine 2,3-dioxygenase mRNA in CD33+ MDSCs derived from human peripheral blood mononuclear cells. These results suggest a dual role for intra-tumorally injected CHST15 siRNA on modulating the tumor immune microenvironment for T cell entry and remotely diminishing CHST15+ MDSCs, decreasing T cell suppression and expanding T cells in the TDLN, ultimately leading to an enhanced accumulation of TILs.

Abstract The combination of mass spectrometry and single crystal X‐ray diffraction of HPLC‐purified DNA‐stabilized silver nanoclusters (DNA‐AgNCs) is a powerful tool to determine the charge and structure of the encapsulated AgNC. Such information is not only relevant to design new DNA‐AgNCs with tailored properties, but it is also important for bio‐conjugation experiments and is essential for electronic structure calculations. Here, the efforts to determine the structure of a HPLC‐purified green emissive DNA‐AgNC are presented. Unfortunately, the original DNA‐AgNC, known to have four valence electrons, could not be crystallized. By modifying the stabilizing DNA sequence, while maintaining the original spectroscopic properties, several mutants could be successfully crystallized, and for one of them, single crystal X‐ray diffraction data provided insight into the silver positions. While the DNA conformation is not resolved, the described approach provides valuable insight into the class of green and dual emissive DNA‐AgNCs with four valence electrons. These results constitute a roadmap on how to improve crystallization and crystal quality for X‐ray diffraction measurements.

Numerous applications of metal-mediated base pairs (metallo-base-pairs) to nucleic acid based nanodevices and genetic code expansion have been extensively studied. Many of these metallo-base-pairs are formed in DNA and RNA duplexes containing Watson-Crick base pairs. Recently, a crystal structure of a metal-DNA nanowire with an uninterrupted one-dimensional silver array was reported. We now report the crystal structure of a novel DNA helical wire containing HgII -mediated T:T and T:G base pairs and water-mediated C:C base pairs. The Hg-DNA wire does not contain any Watson-Crick base pairs. Crystals of the Hg-DNA wire, which is the first DNA wire structure driven by HgII ions, were obtained by mixing the short oligonucleotide d(TTTGC) and HgII ions. This study demonstrates the potential of metallo-DNA to form various structural components that can be used for functional nanodevices.