Genetically Encoded RNA Sensors for Ratiometric and Multiplexed Imaging of Small Molecules in Living Cells.

Genetically encoded sensors afford powerful tools for studying small molecules and metabolites in live cells. However, genetically encoded sensors with a general design remain to be developed. Here we develop genetically encoded RNA sensors with a modular design for ratiometric and multiplexed imaging of small molecules in live cells. The sensor utilizes aptazyme as a recognition module and the light-up RNA aptamer as a signal reporter. The conformation of light-up aptamers is abrogated by a blocking sequence, and aptazyme-mediated cleavage restores the correct conformation, delivering activated fluorescence for small molecule imaging. We first developed a genetically encoded ratiometric sensor using Mango aptamer as a reference and SRB2 as a reporter. It is shown that the sensor allows quantitative imaging and detection of theophylline in live cells. The generality of the design is further demonstrated for imaging other small molecules by replacing the aptazymes. Its ability for multiplexed imaging of small molecules is further explored via the integration of different small-molecule responsive aptazymes and light-up RNA aptamers. This modular design could offer a versatile platform for imaging diverse molecules in living cells.

Genetically Encoded Light-Up RNA Amplifier Dissecting MicroRNA Activity in Live Cells.

Live cell dissection of microRNA activities is crucial for basic and translational medicine, but current hybridization-based strategies may fail to dissect surrounding-dependent activities. Here, we develop a genetically encoded miRNA-induced light-up RNA amplifier (iLAMP) that enables fast-activated, signal-amplified, fluorogenic imaging of miRNA activities in live cells. iLAMP responds to miRNA targets in the mode of "activation upon cleavage", in which the light-up RNA aptamer restores its fluorescence rapidly upon cleavage by the RNA-induced silencing complex. We demonstrate that iLAMP affords substantial signal amplification of ∼100-fold and high specificity in single nucleotide discrimination because of the miRNA-mediated cyclic cleavage. Combined with a Mango RNA aptamer reference module and a pseudoknot terminal stabilizer, iLAMP is shown for quantitative ratiometric imaging and dynamic monitoring of miRNA activities under exogenous stimulations. iLAMP is featured by a modular "plug and play" design and can be readily adapted to the detection of other miRNAs, highlighting its potential in tracking cell differentiation and screening miRNA therapeutics.

Genetically Encoded Dual-Color Light-Up RNA Sensor Enabled Ratiometric Imaging of MicroRNA.

MicroRNAs (miRNAs) play essential roles in regulating gene expression and cell fate. However, it remains a great challenge to image miRNAs with high accuracy in living cells. Here, we report a novel genetically encoded dual-color light-up RNA sensor for ratiometric imaging of miRNAs using Mango as an internal reference and SRB2 as the sensor module. This genetically encoded sensor is designed by expressing a splittable fusion of the internal reference and sensor module under a single promoter. This design strategy allows synchronous expression of the two modules with negligible interference. Live cell imaging studies reveal that the genetically encoded ratiometric RNA sensor responds specifically to mir-224. Moreover, the sensor-to-Mango fluorescence ratios are linearly correlated with the concentrations of mir-224, confirming their capability of determining mir-224 concentrations in living cells. Our genetically encoded light-up RNA sensor also enables ratiometric imaging of mir-224 in different cell lines. This strategy could provide a versatile approach for ratiometric imaging of intracellular RNAs, affording powerful tools for interrogating RNA functions and abundance in living cells.

[Investigation of metabolism of HN-1 isolated from Millettia pachyloba in vivo and in vitro].

Ultra-fast performance liquid chromatography-mass spectrometry( UFLC-MS/MS) was used to study the anti-inflammatory active ingredient of Millettia pachyloba,6-methoxy-8,8-dimethyl-3-( 2,4,5-trimethoxyphenyl)-4 H,8 H-pyrano[2,3-f]chromen-4-one( HN-1),in liver microsomes of rats,mice,rhesus monkeys,Beagle dogs and humans metabolic stability,and compare the metabolic differences between different species. The metabolic phenotype in human liver microsomes was determined by chemical inhibitor method. Using UPLC-Q-TOF-MS/MS detection method,the in vitro metabolites of various liver microsomes were preliminarily inferred by comparing the samples incubated for 0 min and 60 min in vitro. The metabolites of HN-1 in SD rats were presumed by comparing feces,urine,plasma blanks and samples after administration. The results showed that the metabolism of HN-1 in various liver microsomes was stable,and the metabolic properties of dog and human liver microsomes were the closest. It is mainly catabolized by CYP1 A1,CYP2 D6 and CYP3 A4 isoenzymes in human liver microsomes. The metabolites of HN-1 in vitro and in vivo,including 3 in vitro metabolites and5 in vivo metabolites,were preliminarily estimated. The results laid the foundation for further pharmacological studies of HN-1.

Cell Surface-Anchored DNA Nanomachine for Dynamically Tunable Sensing and Imaging of Extracellular pH.

DNA nanodevices that mimic natural biomolecular machines changing configurations in response to external inputs have enabled smart sensors to live cell imaging. We report for the first time the development of a dynamic DNA nanomachine that is anchored on a cell's surface and undergoes pH-responsive triplex-duplex conformation switching, allowing tunable sensing and imaging of extracellular pH. Results reveal that the DNA nanomachine can be stably anchored on the cell surface via multiple anchors, and the adjustment of C+G-C content in the switch element confers tunability of pH response windows. The anchored DNA nanomachine also demonstrates desirable sensitivity, excellent reversibility, and quantitative ability for extracellular pH detection and imaging. This cell surface-anchored pH-responsive DNA nanomachine can provide a useful platform for pH sensing in extracellular microenvironments and diagnostics of different pH-related diseases.