Site-Specific Covalent Conjugation of Modified mRNA by tRNA Guanine Transglycosylase.

Modified mRNA (mod-mRNA) has recently been widely studied as the form of RNA useful for therapeutic applications due to its high stability and lowered immune response. Herein, we extend the scope of the recently established RNA-TAG (transglycosylation at guanosine) methodology, a novel approach for genetically encoded site-specific labeling of large mRNA transcripts, by employing mod-mRNA as substrate. As a proof of concept, we covalently attached a fluorescent probe to mCherry encoding mod-mRNA transcripts bearing 5-methylcytidine and/or pseudouridine substitutions with high labeling efficiencies. To provide a versatile labeling methodology with a wide range of possible applications, we employed a two-step strategy for functionalization of the mod-mRNA to highlight the therapeutic potential of this new methodology. We envision that this novel and facile labeling methodology of mod-RNA will have great potential in decorating both coding and noncoding therapeutic RNAs with a variety of diagnostic and functional moieties.

Light-Activated Control of Translation by Enzymatic Covalent mRNA Labeling.

Activation of cellular protein expression upon visible-light photocleavage of small-molecule caging groups covalently attached to the 5' untranslated region (5' UTR) of an mRNA was achieved. These photocleavable caging groups are conjugated to in vitro transcribed mRNA (IVT-mRNA) through RNA transglycosylation, an enzymatic process in which a bacterial tRNA guanine transglycosylase (TGT) exchanges a guanine nucleobase in a specific 17-nucleotide motif (Tag) for synthetic pre-queuosine1 (preQ1 ) derivatives. The caging groups severely reduce mRNA translation efficiency when strategically placed in the 5' UTR. Using this method, we demonstrate the successful spatiotemporal photoregulation of gene expression with single-cell precision. Our method can be applied to therapeutically relevant chemically modified mRNA (mod-mRNA) transcripts. This strategy provides a modular and efficient approach for developing synthetic gene regulatory circuits, biotechnological applications, and therapeutic discovery.

Site-Specific Covalent Labeling of RNA by Enzymatic Transglycosylation.

We demonstrate the site-specific incorporation of nucleobase derivatives bearing fluorophores or affinity labels into a short RNA stem loop recognition motif by exchange of a guanine residue. The RNA-TAG (transglycosylation at guanosine) is carried out by a bacterial (E. coli) tRNA guanine transglycosylase (TGT), whose natural substrate is the nitrogenous base PreQ1. Remarkably, we have successfully incorporated large functional groups including biotin, BODIPY, thiazole orange, and Cy7 through a polyethylene glycol linker attached to the exocyclic amine of PreQ1. Larger RNAs, such as mRNA transcripts, can be site-specifically labeled if they possess the 17-nucleotide hairpin recognition motif. The RNA-TAG methodology could facilitate the detection and manipulation of RNA molecules by enabling the direct incorporation of functional artificial nucleobases using a simple hairpin recognition element.

Fluorescent turn-on probes for wash-free mRNA imaging via covalent site-specific enzymatic labeling.

Investigating the many roles RNA plays in cellular regulation and function has increased demand for tools to explore RNA tracking and localization within cells. Our recently reported RNA-TAG (transglycosylation at guanine) approach uses an RNA-modifying enzyme, tRNA-guanine transglycosylase (TGT), to accomplish covalent labeling of an RNA of interest with fluorescent tracking agents in a highly selective and efficient manner. Unfortunately, labeling by this method currently suffers from a high nonspecific fluorescent background and is currently unsuitable for imaging RNA within complex cellular environments. Herein we report the design and synthesis of novel fluorogenic thiazole orange probes that significantly lower nonspecific binding and background fluorescence and, as a result, provide up to a 100-fold fluorescence intensity increase after labeling. Using these fluorogenic labeling agents, we were able to image mRNA expressed in Chinese Hamster Ovary cells in a wash-free manner.

[Characteristics and Evaluation of Heavy Metal Pollution in Vegetables in Guangzhou].

Vegetable is an indispensible component of human daily diet,and contamination of vegetables by heavy metals directly threatens human health.In this study,116 vegetable samples were collected from 12 administrative districts of Guangzhou City for analysis of six heavy metals,Cu,Zn,Pb,Cd,Ni,Cr.A combination of single factor evaluation and Nemero Index analysis was employed to determine specific heavy metals exceeding allowable standards and analyze the characteristics of pollution.Risk of exposure was utilized to assess human health risks originating from eating locally planted vegetables contaminated by heavy metals.The results showed that contents of Cu,Zn in the 8 sorts of vegetables were below the standards of maximum allowable content and the contents of heavy mental Cr of up to 91.67% vegetable samples were higher than their standard.Lettuce sativa var.angustana Irish,Luffa acutangula L.,Lycopersicon esculentum Mill.and Daucus carota L.were the 4 species of Pb exceeding vegetables,with the exceed ratio reaching up to 35.71% and Daucus carota L.exceeded the target value most seriously.Only the content of Cd in Lycopersicon esculentum Mill.was over-standard,with the rate of 31.25%.And the highest rate of over-standard of the content of Ni in 3 species of vegetables,which included Lactuca sativa L.,Ipomoea aquatica Forsk and Brassica parachinensis,reached 8.33%.For the contamination level of the eight kinds of vegetable,Lactuca sativa L.,Ipomoea aquatica Forsk,Brassica parachinensis,Raphanus sativus L.and Daucus carota L.were put into the class of alarming,while Lettuce sativa var.angustana Irish,Luffa acutangula L.and Lycopersicon esculentum Mill.were classified as secure.Heavy metals' comprehensive pollution degree of 4 species of vegetables presented a trend of leafy vegetables >rootstalk vegetables >stem vegetables >solanaceous fruits.Health risk assessment showed that Guangzhou citizens eat more frequently Ipomoea aquatica Forsk and Lactuca sativa L.and Brassica parachinensis were prone to higher accumulation of heavy metals,and the dietary intake of heavy metal Cr might cause harm to human health and intake of Cd would bring potential health risk to the human body.Risk of exposure to heavy metal through oral ingestion of vegetables was proved to be higher for children than adults.

Rapid access to phospholipid analogs using thiol-yne chemistry.

Phospholipids and glycolipids constitute an essential part of biological membranes, and are of tremendous fundamental and practical interest. Unfortunately, the preparation of functional phospholipids, or synthetic analogs, is often synthetically challenging. Here we utilize thiol-yne click chemistry methodology to gain access to phospho- and glycolipid analogs. Alkynyl hydrophilic head groups readily photoreact with numerous thiol modified lipid tails to yield the appropriate dithioether phospho- or glycolipids. The resulting structures closely resemble the structure and function of native diacylglycerolipids. Dithioether phosphatidylcholines (PCs) are suitable for forming giant unilamellar vesicles (GUV), which can be used as vessels for cell-free expression systems. The unnatural thioether linkages render the lipids resistant to phospholipase A2 hydrolysis. We utilize the improved stability of these lipids to control the shrinkage of GUVs composed of a mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and dioleyl-dithioether PC, concentrating encapsulated nanoparticles. We imagine that these readily accessible lipids could find a number of applications as natural lipid substitutes.