A universal orthogonal imaging platform for living-cell RNA detection using fluorogenic RNA aptamers.

MicroRNAs (miRNAs) are crucial regulators of gene expression at the post-transcriptional level, offering valuable insights into disease mechanisms and prospects for targeted therapeutic interventions. Herein, we present a class of miRNA-induced light-up RNA sensors (miLS) that are founded on the toehold mediated principle and employ the fluorogenic RNA aptamers Pepper and Squash as imaging modules. By incorporating a sensor switch to disrupt the stabilizing stem of these aptamers, our design offers enhanced flexibility and convertibility for different target miRNAs and aptamers. These sensors detect multiple miRNA targets (miR-21 and miR-122) with detection limits of 0.48 and 0.2 nM, respectively, while achieving a robust signal-to-noise ratio of up to 44 times. Capitalizing on the distinct fluorescence imaging channels afforded by Pepper-HBC620 (red) and Squash-DFHBI-1T (green), we establish an orthogonal miRNA activation imaging platform, enabling the simultaneous visualization of different intracellular miRNAs in living cells. Our dual-color orthogonal miLS imaging platform provides a powerful tool for sequence-specific miRNA imaging in different cells, opening up new avenues for studying the intricate functions of RNA in living cells.

Engineering fluorescent protein chromophores with an internal reference for high-fidelity ratiometric G4 imaging in living cells.

G-quadruplexes (G4s) are significant nucleic acid secondary structures formed by guanine-rich sequences. Many single-emission G4 fluorescent probes that are lit up by inhibiting intramolecular rotation have been reported. However, they are non-fluorescent unless structurally rigidified, making them sensitive to other intracellular crowding and confinement environments in the cell, like viscosity. Ratiometric measurements provide built-in self-calibration for signal correction, enabling more sensitive and reliable detection. Herein, we structurally modulate green fluorescent protein (GFP)-like chromophores by integrating the imidazolidinone scaffold of the GFP chromophore and coumarin 6H, obtaining a G4 responsive dual-emission chromophore, called NHCouI. The red emission signal of NHCouI can specifically respond to parallel G4s, while its green emission signal is inert and acts as an internal reference signal. NHCouI-G4 complexes feature high fluorescence quantum yield and excellent anti-photobleaching properties. NHCouI can self-calibrate the signal and avoid viscosity disturbances within the range of major subcellular organelles during G4 imaging in living cells. It is also applied to reflect the difference between apoptosis and ferroptosis via tracking G4s. To the best of our knowledge, NHCouI is the first small molecule G4 probe enabled by internal reference correction capability, opening up new avenues for dual-emission chromophore development and high-fidelity and reliable analysis in G4 imaging research.

Advances in G-quadruplexes-based fluorescent imaging.

G-quadruplexes (G4s), the noncanonical nucleic acid secondary structure, form within guanine-rich DNA or RNA sequences. G4s formation can affect chromatin architecture and gene regulation and has been associated with various cellular functions, including DNA replication, transcription, and genome maintenance. Visualizing and detecting G4s precisely in such processes is essential to increasing our understanding of G4s biology. Considerable attention has focused on the G4s targeting molecular imaging studies. Besides, fluorescent light-up aptamers (FLAPs, also referred to as fluorogenic aptamers) have gained momentum, which commonly have a G4 scaffolding for imaging intracellular RNAs and metabolites. In this review, we first introduce several representative fluorescent imaging approaches for tracking G4s in cells and in vivo. We also discuss the potential of G4-containing FLAPs in bioimaging and summarize current developments in this field from the standpoint of fluorescent molecules. Finally, we discuss the present challenges and future potential of G4 imaging and G4-containing FLAPs development.

[Effect of Activated Carbon Addition on the Anaerobic Fermentation of Corn Straw in Mesophilic and Thermophilic Conditions].

In order to improve the methane production and concentration, effect of activated carbon addition on the anaerobic fermentation of corn straw under the conditions of mesophilic temperature (38℃) and thermophilic temperature(50℃) was investigated in this study. The results showed that the addition of activated carbon could significantly promote methane production. Compared with the control group in mesophilic and thermophilic conditions, cumulative methane production could be increased by 63% and 96% in test groups. By DGGE analysis, the bacterium enriched by addition of activated carbon was mainly Clostridiales bacterium, compared to Bacillus (without adding activated carbon) in the thermophilic system, while the differences in fermentation with adding activated carbon and without adding activated carbon was not significant in the mesophilic system. With addition of activated carbon, the archaea enriched in the fermentation liquid was mainly Methanosaeta concilii in the mesophilic system, whereas the archaea enriched in the fermentation liquid was mainly Methanosarcina acetivorans in the thermophilic system. The archaea enriched on activated carbon was mainly Methanosaeta concilii at mesophilic temperature, while the archaea enriched on activated carbon was mainly Methanosarcina thermophila at thermophilic temperature.