Imaging the dynamics of messenger RNA with a bright and stable green fluorescent RNA.

Fluorescent RNAs (FRs) provide an attractive approach to visualizing RNAs in live cells. Although the color palette of FRs has been greatly expanded recently, a green FR with high cellular brightness and photostability is still highly desired. Here we develop a fluorogenic RNA aptamer, termed Okra, that can bind and activate the fluorophore ligand ACE to emit bright green fluorescence. Okra has an order of magnitude enhanced cellular brightness than currently available green FRs, allowing the robust imaging of messenger RNA in both live bacterial and mammalian cells. We further demonstrate the usefulness of Okra for time-resolved measurements of ACTB mRNA trafficking to stress granules, as well as live-cell dual-color superresolution imaging of RNA in combination with Pepper620, revealing nonuniform and distinct distributions of different RNAs throughout the granules. The favorable properties of Okra make it a versatile tool for the study of RNA dynamics and subcellular localization.

Large Stokes shift fluorescent RNAs for dual-emission fluorescence and bioluminescence imaging in live cells.

Fluorescent RNAs, aptamers that bind and activate small fluorogenic dyes, have provided a particularly attractive approach to visualizing RNAs in live cells. However, the simultaneous imaging of multiple RNAs remains challenging due to a lack of bright and stable fluorescent RNAs with bio-orthogonality and suitable spectral properties. Here, we develop the Clivias, a series of small, monomeric and stable orange-to-red fluorescent RNAs with large Stokes shifts of up to 108 nm, enabling the simple and robust imaging of RNA with minimal perturbation of the target RNA's localization and functionality. In combination with Pepper fluorescent RNAs, the Clivias enable the single-excitation two-emission dual-color imaging of cellular RNAs and genomic loci. Clivias can also be used to detect RNA-protein interactions by bioluminescent imaging both in live cells and in vivo. We believe that these large Stokes shift fluorescent RNAs will be useful tools for the tracking and quantification of multiple RNAs in diverse biological processes.

Diagnostic Efficacy and Possible Underlying Mechanisms of Noninvasive Clinical Markers in Hepatocellular Carcinoma.

Background and Aims: In this study, we aimed to evaluate the diagnostic values of alpha-fetoprotein (AFP), soluble AXL (sAXL), des-γ-carboxy prothrombin (DCP), the aspartate aminotransferase-to-platelet ratio index (APRI), and the gamma-glutamyl transpeptidase-to-platelet ratio (GPR) in hepatocellular carcinoma (HCC) and the possible underlying mechanisms of the correlations between them. Methods: We collected serum samples from 190, 128, and 75 patients with HCC, cirrhosis, and chronic viral hepatitis, and from 82 healthy subjects. Serum levels of AFP, sAXL, and DCP were determined, and APRI and GPR values were calculated. Receiver operating characteristic (ROC) curves were used to analyze the diagnostic value of single and combined biomarkers. Results: We detected significant differences between the HCC group and other groups regarding serum AFP, sAXL, DCP, and APRI levels. GPR significantly differed between the HCC group and other groups, except for the liver cirrhosis group. AFP, sAXL, DCP, APRI, and GPR had positive correlations with each other, and AFP showed a higher area under the curve (AUC) and Youden index values, while APRI and DCP showed the highest sensitivity and specificity. Also, when AFP was combined with sAXL, DCP, APRI, and GRP, the highest AUC (0.911) and a higher net reclassification improvement value were obtained compared with those obtained for the individual biomarkers. Conclusions: AFP, sAXL, DCP, APRI, and GPR are independent risk factors for HCC, and the diagnostic performance of AFP combined with sAXL, DCP, APRI, and GPR for HCC diagnosis was superior to that of the individual biomarkers.

Design of a palette of SNAP-tag mimics of fluorescent proteins and their use as cell reporters.

Naturally occurring fluorescent proteins (FPs) are the most widely used tools for tracking cellular proteins and sensing cellular events. Here, we chemically evolved the self-labeling SNAP-tag into a palette of SNAP-tag mimics of fluorescent proteins (SmFPs) that possess bright, rapidly inducible fluorescence ranging from cyan to infrared. SmFPs are integral chemical-genetic entities based on the same fluorogenic principle as FPs, i.e., induction of fluorescence of non-emitting molecular rotors by conformational locking. We demonstrate the usefulness of these SmFPs in real-time tracking of protein expression, degradation, binding interactions, trafficking, and assembly, and show that these optimally designed SmFPs outperform FPs like GFP in many important ways. We further show that the fluorescence of circularly permuted SmFPs is sensitive to the conformational changes of their fusion partners, and that these fusion partners can be used for the development of single SmFP-based genetically encoded calcium sensors for live cell imaging.

Visible-light activatable coumarin-based phototriggers for fluorescence imaging with ultra-high photolysis efficiency.

Photoactivated fluorophores (PAFs) are powerful imaging tools for observing subcellular structures and tracking dynamic biological processes. However, photoremovable protecting groups (PPGs) widely used to construct PAFs suffer from the drawbacks of short-wavelength excitation and/or low photolysis efficiency. Herein, a class of coumarin-based PPGs with electron-rich thiophene derived substitutions at the C3-position of a coumarin scaffold were prepared. The modification not only leads to the redshift of the absorption band to the blue light region (400-500 nm), but also the increases of uncaging quantum yield (Φu) as well as molar extinction coefficient (εmax), thus enhancing the photolysis efficiency (Φu × Îµmax) up to 34.2 × 103 M-1 cm-1. The exceptionally high photolysis efficiency enables efficient photolysis in blue light as weak as 2 mW cm-2 or in blue light from a Luminol chemiluminescence system. Based on the excellent photolysis properties, the PAF constructed by the new PPG exhibits fast photoactivation and a low background signal, and the resulting fluorescence images display a signal-to-noise ratio greater than 780. It is anticipated that the superior photolysis performance makes the PPGs a novel platform for the construction of photo responsive systems in a variety of applications.

Improved myocardial performance in infarcted rat heart by injection of disulfide-cross-linked chitosan hydrogels loaded with basic fibroblast growth factor.

Myocardial infarction (MI) has been considered as the leading cause of cardiovascular-related deaths worldwide. Basic fibroblast growth factor (bFGF) is a member of the fibroblast growth factor family that promotes angiogenesis after MI; however, it has poor clinical efficacy due to proteolytic degradation, low drug accumulation, and severe drug-induced side effects. In this study, an injectable disulfide-cross-linked chitosan hydrogel loaded with bFGF was prepared via a thiol-disulfide exchange reaction for MI treatment. The thiol-disulfide exchange reaction between pyridyl disulfide-modified carboxymethyl chitosan (CMCS-S-S-Py) and reduced BSA (rBSA) was carried out under physiological conditions (37 °C and pH 7.4). The mechanical properties of the disulfide-cross-linked chitosan hydrogel were evaluated based on the molar ratio of the pyridyl disulfide groups of CMCS-S-S-Py and the thiol groups of rBSA. The disulfide-cross-linked chitosan hydrogel showed good swelling performance, rapid glutathione-triggered degradation behavior and well-defined cell proliferation towards NIH 3T3 fibroblast cells. In the process of establishing a rat MI model, the squeezing heart method was used to make the operation more accurate and the mortality of rats was decreased by using a ventilator. The disulfide-cross-linked chitosan hydrogel loaded with bFGF (bFGF-hydrogel) was injected into a peri-infarcted area of cardiac tissue immediately following MI. Echocardiography demonstrated that the left ventricular functions were improved by the bFGF-hydrogel after 28 days of treatment. Histological results revealed that the hydrogel significantly reduced the fibrotic area of MI, and this was further improved by the bFGF-hydrogel treatment. TUNEL and immunohistochemical staining results showed that the bFGF-hydrogel had a more synergistic effect on antiapoptosis and proangiogenesis than using either bFGF or the hydrogel alone.

Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs.

Fluorescent RNAs (FRs), aptamers that bind and activate fluorescent dyes, have been used to image abundant cellular RNA species. However, limitations such as low brightness and limited availability of dye/aptamer combinations with different spectral characteristics have limited use of these tools in live mammalian cells and in vivo. Here, we develop Peppers, a series of monomeric, bright and stable FRs with a broad range of emission maxima spanning from cyan to red. Peppers allow simple and robust imaging of diverse RNA species in live cells with minimal perturbation of the target RNA's transcription, localization and translation. Quantification of the levels of proteins and their messenger RNAs in single cells suggests that translation is governed by normal enzyme kinetics but with marked heterogeneity. We further show that Peppers can be used for imaging genomic loci with CRISPR display, for real-time tracking of protein-RNA tethering, and for super-resolution imaging. We believe these FRs will be useful tools for live imaging of cellular RNAs.