Site-specific RNA modification via initiation of in vitro transcription reactions with m6A and isomorphic emissive adenosine analogs.

The templated enzymatic incorporation of adenosine and its analogs, including m6A, thA and tzA into RNA transcripts, has been explored. Enforced transcription initiation with excess free nucleosides and the native triphosphates generates 5'-end modified transcripts, which can be 5'-phosphorylated and ligated to provide full length, singly modified RNA oligomers. To explore structural integrity, functionality and utility of the resulting non-canonical purine-containing RNA constructs, a MazF RNA hairpin substrate has been synthesized and analyzed for its susceptibility to this endonuclease. Additionally, RNA substrates, containing a singly incorporated isomorphic emissive nucleoside, can be used to monitor the enzymatic reactions in real-time by steady state fluorescence spectroscopy.

Isomorphic Fluorescent Nucleosides Facilitate Real-Time Monitoring of RNA Depurination by Ribosome Inactivating Proteins.

Ribosome-inactivating proteins, a family of highly cytotoxic proteins, interfere with protein synthesis by depurinating a specific adenosine residue within the conserved α-sarcin/ricin loop of eukaryotic ribosomal RNA. Besides being biological warfare agents, certain RIPs have been promoted as potential therapeutic tools. Monitoring their deglycosylation activity and their inhibition in real time have remained, however, elusive. Herein, we describe the enzymatic preparation and utility of consensus RIP hairpin substrates in which specific G residues, next to the depurination site, are surgically replaced with tz G and th G, fluorescent G analogs. By strategically modifying key positions with responsive fluorescent surrogate nucleotides, RIP-mediated depurination can be monitored in real time by steady-state fluorescence spectroscopy. Subtle differences observed in preferential depurination sites provide insight into the RNA folding as well as RIPs' substrate recognition features.

Dual-emissive nanoarchitecture of lanthanide-complex-modified silica particles for in vivo ratiometric time-gated luminescence imaging of hypochlorous acid.

We have developed a ratiometric time-gated luminescence sensory system for in vivo imaging of hypochlorous acid (HClO) by preparing a dual-emissive nanoarchitecture of europium- and terbium-complex-modified silica nanoparticles. The design of this nanoarchitecture is based on our new finding that the strong, long-lived luminescence of the ß-diketonate-Eu3+ complex can be rapidly and selectively quenched by HClO. Therefore, the ß-diketonate-Eu3+ complex was decorated on the surface of the silica nanoparticles for responding to HClO, while a HClO-insensitive luminescent terbium complex was immobilized in the inner solid core of the nanoparticles to serve as an internal standard. This nanosensing probe combines the advantages of both ratiometric and time-gated detection modes to afford high accuracy and sensitivity. Upon exposure to HClO, the nanoprobe displayed a remarkable luminescence color change from red to green, and the intensity ratio of the green over the red luminescence (I539/I607) showed a rapid, sensitive and selective response to HClO. Additionally, the feasibility of using the nanoprobe for intracellular detection of exogenous and endogenous HClO and for real-time mapping of HClO in small laboratory animals has been demonstrated via ratiometric time-gated luminescence imaging microscopy. The results reveal that the constructed nanoarchitecture cloud is a favorable and useful sensing probe for the real-time imaging of HClO in vivo with high specificity and contrast.