Tetrazine-Induced Bioorthogonal Activation of Vitamin E-Modified siRNA for Gene Silencing

The temporal activation of siRNA provides a valuable strategy for the regulation of siRNA activity and conditional gene silencing. The bioorthogonal bond-cleavage reaction of benzonorbonadiene and tetrazine is a promising trigger in siRNA temporal activation. Here, we developed a new method for the bio-orthogonal chemical activation of siRNA based on the tetrazine-induced bond-cleavage reaction. Small-molecule activatable caged siRNAs were developed with the 5′-vitamin E-benzonobonadiene-modified antisense strand targeting the green fluorescent protein (GFP) gene and the mitotic kinesin-5 (Eg5) gene. The addition of tetrazine triggered the reaction with benzonobonadiene linker and induced the linker cleavage to release the active siRNA. Additionally, the conditional gene silencing of both exogenous GFP and endogenous Eg5 genes was successfully achieved with 5′-vitamin E-benzonobonadiene-caged siRNAs, which provides a new uncaging strategy with small molecules

Circular siRNAs for Reducing Off-Target Effects and Enhancing Long-Term Gene Silencing in Cells and Mice

Circular non-coding RNAs are found to play important roles in biology but are still relatively unexplored as a structural motif for chemically regulating gene function. Here, we investigated whether small interfering RNA (siRNA) with a circular structure can circumvent off-target gene silencing, a problem often observed with standard linear duplex siRNA. In the present work, we, for the first time, synthesized a series of circular siRNAs by cyclizing two ends of a single-stranded RNA (sense or antisense strand) to construct circular siRNAs that were more resistant to enzymatic degradation. Gene silencing of GFP and luciferase was successfully achieved using these circular siRNAs with circular sense strand RNAs and their complementary linear antisense strand RNAs. The off-target effect of sense strand RNAs was evaluated and no cross off-target effects were observed. In addition, we successfully achieved longer gene-silencing efficiency in mice with circular siRNAs than with linear siRNAs. These results indicate the promise of circular siRNAs for overcoming off-target effects of siRNAs and enhancing the possible long-term effect of siRNA gene silencing in basic research and drug development

Application of X-ray computed microtomography (microCT) to the study of larger foraminifera

FTO demethylates internal N-6-methyladenosine (m(6)A) and N-6,2'-O-dimethyladenosine (m(6)A(m); at the cap + 1 position) in mRNA, m(6)A and m(6)A(m) in snRNA, and N-1-methyladenosine (m(1)A) in tRNA in vivo, and in vitro evidence supports that it can also demethylate N-6-methyldeoxyadenosine ((6)mA), 3-methylthymine (3mT), and 3-methyluracil (m(3)U). However, it remains unclear how FTO variously recognizes and catalyzes these diverse substrates. Here we demonstrate-in vitro and in vivo-that FTO has extensive demethylation enzymatic activity on both internal m(6)A and cap m(6)A(m). Considering that (6)mA, m(6)A, and m(6)A(m) all share the same nucleobase, we present a crystal structure of human FTO bound to (6)mA-modified ssDNA, revealing the molecular basis of the catalytic demethylation of FTO toward multiple RNA substrates. We discovered that (i) N-6-methyladenine is the most favorable nucleobase substrate of FTO, (ii) FTO displays the same demethylation activity toward internal m(6)A andm(6)A(m) in the same RNA sequence, suggesting that the substrate specificity of FTO primarily results from the interaction of residues in the catalytic pocket with the nucleobase (rather than the ribose ring), and (iii) the sequence and the tertiary structure of RNA can affect the catalytic activity of FTO. Our findings provide a structural basis for understanding the catalytic mechanism through which FTO demethylates its multiple substrates and pave the way forward for the structure-guided design of selective chemicals for functional studies and potential therapeutic applications

A bimolecular modification strategy for developing long-lasting bone anabolic aptamer

The molecular weight of nucleic acid aptamers (20 kDa) is lower than the cutoff threshold of the renal filtration (30–50 kDa), resulting in a very short half-life, which dramatically limits their druggability. To address this, we utilized 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(4-hydroxy-2-oxo-2H-chromen-6-yl)propenamide (HC) and 12-((2,5-dioxopyrrolidin-1-yl)oxy)-12-oxododecanoic acid (DA), two newly designed coupling agents, for synergistic binding to human serum albumin (HSA). Both HC and DA are conjugated to a bone anabolic aptamer (Apc001) against sclerostin to form an Apc001OC conjugate with high binding affinity to HSA. Notably, HC and DA could synergistically facilitate prolonging the half-life of the conjugated Apc001 and promoting its bone anabolic potential. Using the designed blocking peptides, the mechanism studies indicate that the synergistic effect of HC-DA on pharmacokinetics and bone anabolic potential of the conjugated Apc001 is achieved via their synergistic binding to HSA. Moreover, biweekly Apc001OC at 50 mg/kg shows comparable bone anabolic potential to the marketed sclerostin antibody given weekly at 25 mg/kg. This proposed bimolecular modification strategy could help address the druggability challenge for aptamers with a short half-life