Crystallization phasing and obtaining high-quality crystals are bottleneck challenges for the X-ray crystallographic analysis of nucleic acids, especially when dynamic behavior is to be inferred from crystallographic B-factors. Crystallization of DNA duplexes, normally existing in the B-form in solution, is especially challenging, as the high salt used in many crystallization processes favors their transformation to A-form DNA duplexes. To address crystallization challenges while avoiding structural perturbation, we explored atom-specific incorporation to place a selenium atom on the 2′-β (arabino) position of the 2′-deoxyribose ring. This incorporation is expected to favor the B-form of a DNA duplex during crystallization. Here, we report the first synthesis of the β-2′-MeSe-thymidine (SeT) nucleoside, the corresponding Se-phosphoramidite, and Se-containing DNA oligonucleotides. We found that particular Se-DNAs form crystals that are surprisingly larger than we have often observed, having higher quality and giving improved diffraction resolution when compared to crystals from analogous standard oligonucleotides. Surprisingly, one duplex made from a self-complementary Se-containing oligonucleotide gave crystals 600 × 200 μm2 in size; this is 10–100 times larger in volume than the corresponding crystals grown from native DNA. CD and a solved crystal structure showed that the selenium in the β-orientation did not perturb the native structure and gave diffraction data from which dynamic information could be extracted. Crystals of this size are especially important for neutron diffraction studies. Moreover, we discovered that the high-quality Se-DNA crystals diffracted to 1.15 Å. The Se-derivatized structure was virtually identical with the native structure. These discoveries suggest a simple strategy to address other crystallization challenges in nucleic acid crystallography, a strategy whose scope deserves further exploration.

Tellurium was successfully incorporated into proteins and applied to protein structure determination through X-ray crystallography. However, studies on tellurium modification of DNA and RNA are limited. This review highlights the recent development of Te-modified nucleosides, nucleotides, and nucleic acids, and summarizes the main synthetic approaches for the preparation of 5-PhTe, 2′-MeTe, and 2′-PhTe modifications. Those modifications are compatible with solid-phase synthesis and stable during Te-oligonucleotide purification. Moreover, the ideal electronic and atomic properties of tellurium for generating clear isomorphous signals give Te-modified DNA and RNA great potential applications in 3D crystal structure determination through X-ray diffraction. STM study also shows that Te-modified DNA has strong topographic and current peaks, which immediately suggests potential applications in nucleic acid direct imaging, nanomaterials, molecular electronics, and diagnostics. Theoretical studies indicate the potential application of Te-modified nucleosides in cancer therapy.

Chemically modified antisense oligonucleotides (ASO) currently in pre-clinical and clinical experiments mainly focus on the 2'-position derivatizations to enhance stability and targeting affinity. Considering the possible incompatibility of 2'-modifications with RNase H stimulation and activity, we have hypothesized that the atom specific modifications on nucle-obases can retain the complex structure and RNase H activity, while enhancing ASO's binding affinity, specificity, and sta-bility against nucleases. Herein we report a novel strategy to explore our hypothesis by synthesizing the deoxynucleoside phosphoramidite building block with the seleno-modification at 5-position of thymidine, as well as its Se-oligonucleotides. Via X-ray crystal structural study, we found that the Se-modification was located in the major groove of nucleic acid duplex and didn't cause the thermal and structural perturbations. Surprisingly, our nucleobase-modified Se-DNAs were exceptionally resistant to nuclease digestion, while compatible with RNase H activity. This affords a novel avenue for potential anti-sense modification in the form of Se-antisense oligonucleotides (Se-ASO).

Chemically modified antisense oligonucleotides (ASO) currently in pre‐clinical and clinical experiments mainly focus on the 2′‐position derivatizations to enhance stability and targeting affinity. Considering the possible incompatibility of 2′‐modifications with RNase H stimulation and activity, we have hypothesized that the atom specific modifications on nucleobases can retain the complex structure and RNase H activity, while enhancing ASO's binding affinity, specificity, and stability against nucleases. Herein we report a novel strategy to explore our hypothesis by synthesizing the deoxynucleoside phosphoramidite building block with the seleno‐modification at 5‐position of thymidine, as well as its Se‐oligonucleotides. Via X‐ray crystal structural study, we found that the Se‐modification was located in the major groove of nucleic acid duplex and didn't cause the thermal and structural perturbations. Surprisingly, our nucleobase‐modified Se‐DNAs were exceptionally resistant to nuclease digestion, while compatible with RNase H activity. This affords a novel avenue for potential anti‐sense modification in the form of Se‐antisense oligonucleotides (Se‐ASO).