Spatial imaging of glycoRNA in single cells with ARPLA.

Little is known about the biological roles of glycosylated RNAs (glycoRNAs), a recently discovered class of glycosylated molecules, because of a lack of visualization methods. We report sialic acid aptamer and RNA in situ hybridization-mediated proximity ligation assay (ARPLA) to visualize glycoRNAs in single cells with high sensitivity and selectivity. The signal output of ARPLA occurs only when dual recognition of a glycan and an RNA triggers in situ ligation, followed by rolling circle amplification of a complementary DNA, which generates a fluorescent signal by binding fluorophore-labeled oligonucleotides. Using ARPLA, we detect spatial distributions of glycoRNAs on the cell surface and their colocalization with lipid rafts as well as the intracellular trafficking of glycoRNAs through SNARE protein-mediated secretory exocytosis. Studies in breast cell lines suggest that surface glycoRNA is inversely associated with tumor malignancy and metastasis. Investigation of the relationship between glycoRNAs and monocyte-endothelial cell interactions suggests that glycoRNAs may mediate cell-cell interactions during the immune response.

DNAzyme-Based Lithium-Selective Imaging Reveals Higher Lithium Accumulation in Bipolar Disorder Patient-Derived Neurons.

Lithium has been a drug for bipolar disorders (BD) for over 70 years; however, its usage has been limited by its narrow therapeutic window (between 0.6 and 1.2 mM). Understanding the cellular distribution of lithium ions (Li+) in patient cells will offer deep insight into this limitation, but selective imaging of Li+ in living cells under biomedically relevant concentration ranges has not been achieved. Herein, we report in vitro selection and development of a Li+-specific DNAzyme fluorescent sensor with >100-fold selectivity over other biorelevant metal ions. This sensor allows comparative Li+ visualization in HeLa cells, human neuronal progenitor cells (NPCs), and neurons derived from BD patients and healthy controls. Strikingly, we detected enhanced accumulation of Li+ in cells derived from BD patients compared with healthy controls in differentiated neurons but not NPCs. These results establish the DNAzyme-based sensor as a novel platform for biomedical research into BD and related areas using lithium drugs.

PNA-Assisted DNAzymes to Cleave Double-Stranded DNA for Genetic Engineering with High Sequence Fidelity.

DNAzymes have been widely used in many sensing and imaging applications but have rarely been used for genetic engineering since their discovery in 1994, because their substrate scope is mostly limited to single-stranded DNA or RNA, whereas genetic information is stored mostly in double-stranded DNA (dsDNA). To overcome this major limitation, we herein report peptide nucleic acid (PNA)-assisted double-stranded DNA nicking by DNAzymes (PANDA) as the first example to expand DNAzyme activity toward dsDNA. We show that PANDA is programmable in efficiently nicking or causing double strand breaks on target dsDNA, which mimics protein nucleases and can act as restriction enzymes in molecular cloning. In addition to being much smaller than protein enzymes, PANDA has a higher sequence fidelity compared with CRISPR/Cas under the condition we tested, demonstrating its potential as a novel alternative tool for genetic engineering and other biochemical applications.

Regeneration of ß-Carotene from the Radical Cation by Tyrosine and Tryptophan.

The phenolic amino acid tyrosine (Tyr) was found more efficient in regenerating ß-carotene (ß-Car) from the radical cation (ß-Car(•+)) than tryptophan (Trp) in the presence of base for conditions where the reduction potentials for Trp and Tyr are comparable. Electron transfer from Tyr in 4:1 chloroform/methanol to ß-Car(•+) in the presence of excess base, (CH3)4N(+)OH(-), had a rate close to diffusion control and a second-order rate constant in agreement with the Marcus theory for electron transfer when compared to plant phenols. A maximum of 40% ß-Car was regenerated for ten times excess of Tyr as studied by 532 nm laser flash photolysis followed by transient absorption spectroscopy in the visible and near-infrared regions. The nonregenerated fraction of ß-Car is assigned to secondary degradation processes. For Trp, the rate constant for regeneration of ß-Car(•+) was 1 order of magnitude smaller compared to Tyr and slower than expected from Marcus theory by comparison with plant phenols.