Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025097
Some structured RNAs, such as riboswitches and aptamers, can bind to their cognate ligands and have been used in biosensors and gene expression control elements. However, current methods for detecting ligand binding to structured RNAs are either severely limited or inconvenient. In this study, we design a multibase pair bridge to integrate a hammerhead ribozyme into structured RNAs to detect ligand binding events. The experimental results demonstrate that the length of the bridge has a significant effect on the cleavage of the ribozyme; optimal cleavage can be achieved with three to six base pairs in the bridge. The dissociation constant (KD) values obtained through this method are in agreement with those determined by in-line probing techniques, and 1 pmol of allosteric ribozyme RNA is sufficient for measurement. We apply this method to evaluate the binding affinity of the riboswitch candidate Motif_9307. Our findings indicate that this motif has no binding affinity for S-adenosylmethionine or several other tested ligands, which is consistent with the results of the in-line probing experiments. Notably, our method reveals an increase in cleavage activity when yeast extract is added as a mixture of ligands, suggesting that the ligand of Motif_9307 is present in the extract. In conclusion, we develop an alternative approach for measuring ligand binding events associated with riboswitch candidates and aptamers.
Acta Biochimica et Biophysica Sinica•2026•DOI: 10.3724/abbs.2025212
Spatial biology aims to elucidate cellular organization, function, and interactions within native tissue contexts, offering key insights into both normal physiology and disease. Spatial proteomics complements this by enabling high-resolution mapping of protein localization and abundance, directly reflecting functional cellular states. Unlike transcriptomics, which infers potential activity, proteomics captures actual molecular functions, including post-translational modifications and dynamic interactions. However, in situ protein profiling poses significant challenges, as proteins cannot be directly sequenced or easily targeted via nucleic acid hybridization. Antibody-oligonucleotide conjugates (AOCs) address this limitation by converting protein recognition into a DNA-based readout, thereby enabling sensitive and scalable detection. In this review, we outline the core principles of AOC-based spatial proteomic technologies, including multiplexed protein analysis, in situ protein-protein interactions, and integration with other biomolecular data. We highlight their applications in decoding tissue complexity and disease pathology and examine key technical challenges that remain. Overall, AOCs offer distinct advantages, including DNA-mediated signal amplification, spatially resolved proteomic profiling, and compatibility with multi-omics approaches, positioning them as powerful platforms in the advancement of spatial biology.
Acta Biochimica et Biophysica Sinica•2025•DOI: 10.3724/abbs.2025097
Structured RNAs such as riboswitches and aptamers bind cognate ligands and serve as biosensors and gene-control elements, yet existing methods for detecting ligand-binding events are limited or inconvenient. This study designs a multibase pair bridge to integrate a hammerhead ribozyme into structured RNAs, enabling ligand-binding detection via modulation of ribozyme cleavage. Bridge length critically affects cleavage: optimal activity occurs with three to six base pairs. Dissociation constants (KD) obtained by this method agree with in-line probing values, and 1 pmol of allosteric ribozyme RNA suffices for measurement. Applied to riboswitch candidate Motif_9307, the assay revealed no binding affinity for S-adenosylmethionine or several other tested ligands, consistent with in-line probing. Notably, cleavage activity increased upon addition of yeast extract as a ligand mixture, indicating the presence of the Motif_9307 ligand in the extract. This approach provides an alternative method for measuring ligand-binding events associated with riboswitch candidates and aptamers.