TAT-SNAP-23 (University of Louisville)

The application of the dye-labeled fluorescence method in a ligand-RNA interaction assay is a complex and costly process prone to steric hindrance. Fluorescent nanomaterials offer an attractive alternative due to their simple, low-cost synthesis and effective screening properties. Here, CdTe@ZIF-8 core-shell nanocomposites were used as fluorescence signal transducer in the ligand-TAR RNA interaction assay. Different experimental strategies were developed based on the size-selective nature of the CdTe@ZIF-8 nanocomposites. When ligands can quench fluorescence, two assays of fluorescence recovery with TAR RNA and Tat peptide competitive displacement are carried out successively, which can not only distinguish ligands binding to TAR RNA but also screen potential Tat protein antagonists. When ligands cannot quench fluorescence, the mitoxantrone-TAR RNA complex is used in the competitive displacement assay. Ligands that displaced mitoxantrone from the mitoxantrone-TAR RNA complex signaled the interaction with TAR RNA. Eight ligands, including known and unknown TAR RNA-binding ligands, were tested via the above strategies. The results showed that this method was effective at distinguishing the known RNA-binding partner and screening the Tat antagonist from the test ligands. This simple and effective strategy is expected to be suitable for actual drug screening. Graphical abstract.

Alpha B-crystallin (CRYAB) is an important member of the small heat shock protein family, and plays a protective and therapeutic role in neurological inflammation. CRYAB expression was assessed in cultured HT29 and Caco-2 cells and inflamed mucosa of patients with inflammatory bowel disease (IBD) and colitis models in mice. Lentivirus-overexpressing and CRSIPR/Cas9 systems were used in different cells to upregulate and silence CRYAB expression, respectively. Cell permeable recombined fusion protein TAT-CRYAB was injected intraperitoneally into dextran sulfate sodium (DSS)- or 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice to assess its anti-inflammatory effects. CRYAB was found to be significantly decreased in the inflamed mucosa from IBD patients and DSS-induced colitis in mice, and negatively correlated with the levels of TNF-α and IL-6, respectively. Enforced expression of CRYAB suppressed expression of proinflammatory cytokines (e.g., TNF-α, IL-6, IL-1β, and IL-8) via inhibiting the IKK complex formation, whereas lack of CRYAB expression markedly enhanced proinflammatory responses. Consistently, administration of TAT-CRYAB fusion protein significantly alleviated DSS- or TNBS-induced colitis in mice and protected intestinal barrier integrity. CRYAB regulates inflammatory response in intestinal mucosa by inhibiting IKKβ-mediated signaling and may serve as a novel therapeutic approach in the treatment of IBD.