Guzzetti, I. | bioRxiv Science

bioRxiv2024

Biophysics of microRNA-34a targeting and its influence on down-regulation

microRNAs (miRNAs) regulate target mRNA expression post-transcriptionally through their association with Argonaute 2 (AGO2) proteins. Predicting the efficiency of mRNA repression by miRNA has been limited by our comprehension of the structure-function relationship within miRNA binding sites. Using a combination of EMSA, luciferase reporter assays, and structural probing, we investigated the interaction between the human tumour suppressor miR-34a and 12 mRNA targets. Comparison of direct RNA:RNA interactions and those within the functional AGO2 protein revealed that the isolated mRNA:miRNA duplex serves as a strong predictor for duplex affinity and structure within AGO2. Our findings reveal that AGO2 has a bidirectional capacity to modulate affinity; weakening tight RNA:RNA binders while strengthening weak ones. We identified three distinct structural groups that form upon miR-34a binding and reveal a novel structural group that exhibits a guide strand bulge. MD simulations indicate a conceivable fit of this miRNA-bulge structure within AGO2. Our results demonstrate that the structural characteristics of mRNA:miRNA duplexes could serve as contributing determinants of repression efficacy.

biophysics↗

bioRxiv2019

Observing an antisense drug complex in intact human cells by in-cell NMR

Gaining insight into the uptake of drugs into cells, trafficking and their target engagement enhances understanding of the drugs function and efficiency. Here we study an antisense oligonucleotide drug (ASO) delivered into HEK293T and HeLa cells, by Nuclear Magnetic Resonance (NMR). Using a combination of transfection, cryoprotection and dynamic nuclear polarization (DNP), we were able to detect the drug directly in intact frozen cells. Activity of the drug was confirmed by qRT-PCR, measuring downregulation of its target mSTAT3. Applying DNP NMR to frozen cells, we overcome limitations of traditional solution-state in-cell NMR (e.g. size, stability and sensitivity) as well as of visualization techniques, where (e.g. fluorescent) tagging of the ASO decreases its activity. The possibility to study an untagged, active drug, interacting in its natural environment, will increase insights into molecular mechanisms of delivery, intracellular trafficking and target engagement in intact cells.

biophysics↗