Biology subjects

Dantsu, Y.

Publications and source records attributed to Dantsu, Y..

3 recordsLinked to original sources

bioRxiv2024

Selection of Fluorinated Aptamer Targeting RNA Element with Different Chirality

The development of RNA aptamers with high specificity and affinity for target molecules is a critical advancement in the field of therapeutic and diagnostic applications. This study presents the selection of a 2-fluoro modified mirror-image RNA aptamer through the in vitro SELEX process. Using a random RNA library, we performed iterative rounds of selection and amplification to enrich aptamers that bind specifically to the viral frameshift element which contains the opposite chirality. The unnatural chirality of the aptamer improved its enzymatic stability, and the incorporation of 2-fluoro modifications was crucial in enhancing the binding affinity of the aptamers. After nine rounds of SELEX, the enriched RNA pool was sequenced and analyzed, revealing the dominant aptamer sequences. The selected 2-fluoro modified mirror-image RNA aptamer demonstrated a dissociation constant of approximately 1.6 M, indicating moderate binding affinity with the target and exceptional stability against nuclease degradation. Our findings highlight the potential of 2-fluoro modified mirror-image RNA aptamers in enhancing the stability and utility of RNA-based therapeutics and diagnostics, paving the way for future applications in diverse biomedical fields.

biochemistry↗

bioRxiv2023

Insight into the structures of unusual base pairs in RNA complexes containing primer/template/adenosine ligand

In prebiotic RNA world, the self-replication of RNA without enzymes can be achieved through the utilization of 2-aminoimidazole activated nucleotides as efficient substrates. The mechanism of RNA nonenzymatic polymerization has been extensively investigated biophysically and structurally by using the model of RNA primer/template complex which is bound by the imidazolium-bridged or triphosphate-bridged diguanosine intermediate. However, beyond the realm of guanosine substrate, the structural insight into how alternative activated nucleotides bind and interact with RNA primer/template complex remains unexplored, which is important for understanding the low reactivity of adenosine and uridine substrates in RNA primer extension, as well as its relationship with the structures. Here we use crystallography as method and determine a series of high-resolution structures of RNA primer/template complexes bound by ApppG, the close analog of dinucleotide intermediate containing adenosine and guanosine. The structures show that ApppG ligands bind to RNA template through both Watson-Crick and noncanonical base pairs, with the primer 3'-OH group far from the adjacent phosphorus atom of the incoming substrate. The structures indicate that, when adenosine is included in the imidazolium-bridged intermediate, the complexes are likely preorganized in a suboptimal conformation, making it difficult for the primer to in-line attack the substrate. Moreover, by cocrystallizing the RNA primer/template with chemically activated adenosine and guanosine monomers, we successfully observe the slow formation of the imidazolium-bridged intermediate (Ap-AI-pG) and the preorganized structure for RNA primer extension. Overall, our studies offer a structural explanation for the slow rate of RNA primer extension when using adenosine-5-phosphoro-2-aminoimidazolide as a substrate during nonenzymatic polymerization.

biochemistry↗

bioRxiv2023

Syntheses of Pyrimidine-Modified Seleno-DNAs as Stable Antisense Molecules

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 ASOs 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 didnt 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 antisense modification in the form of Se-antisense oli-gonucleotides (Se-ASO).

biochemistry↗