DNAzyme-based faithful probing and pulldown to identify candidate biomarkers of low abundance.

Biomarker discovery is essential for the understanding, diagnosis, targeted therapy and prognosis assessment of malignant diseases. However, it remains a huge challenge due to the lack of sensitive methods to identify disease-specific rare molecules. Here we present MORAC, molecular recognition based on affinity and catalysis, which enables the effective identification of candidate biomarkers with low abundance. MORAC relies on a class of DNAzymes, each cleaving a sole RNA linkage embedded in their DNA chain upon specifically sensing a complex system with no prior knowledge of the system's molecular content. We show that signal amplification from catalysis ensures the DNAzymes high sensitivity (for target probing); meanwhile, a simple RNA-to-DNA mutation can shut down their RNA cleavage ability and turn them into a pure affinity tool (for target pulldown). Using MORAC, we identify previously unknown, low-abundance candidate biomarkers with clear clinical value, including apolipoprotein L6 in breast cancer and seryl-tRNA synthetase 1 in polyps preceding colon cancer.

Mitochondrion-Targeting Fluorescence Probe via Reduction Induced Charge Transfer for Fast Methionine Sulfoxide Reductases Imaging.

Methionine sulfoxide reductases (Msrs) play essential roles in maintaining mitochondrial function and are recognized as potential therapeutic targets. However, current probes for Msrs fail to target mitochondria and exhibit a relatively slow response and limited sensitivity. Here we develop a novel turn-on fluorescence probe that facilitates imaging of mitochondrial Msrs in living cells. The probe is constructed by conjugating a methyl phenyl sulfoxide, a mimic Msrs substrate, to an electron-withdrawing hydrophobic cation, methylpyridinium. The probe of acceptor-acceptor structure is initially nonemissive. Msrs catalyzed reduction of sulfoxide to sulfide generated a fluorophore of distinct donor-acceptor structure. The probe is demonstrated to exhibit high sensitivity, fast response, and high selectivity toward MsrA in vitro. Furthermore, the probe is successfully introduced to detect and image Msrs in living cells with excellent mitochondrial-targeting capability. Moreover, the probe also reveals decreased Msrs activity in a cellular Parkinson's disease model. Our probe affords a powerful tool for detecting and visualizing mitochondrial Msrs in living cells.

Activatable Fluorescence Probe via Self-Immolative Intramolecular Cyclization for Histone Deacetylase Imaging in Live Cells and Tissues.

Histone deacetylases (HDACs) play essential roles in transcription regulation and are valuable theranostic targets. However, there are no activatable fluorescent probes for imaging of HDAC activity in live cells. Here, we develop for the first time a novel activatable two-photon fluorescence probe that enables in situ imaging of HDAC activity in living cells and tissues. The probe is designed by conjugating an acetyl-lysine mimic substrate to a masked aldehyde-containing fluorophore via a cyanoester linker. Upon deacetylation by HDAC, the probe undergoes a rapid self-immolative intramolecular cyclization reaction, producing a cyanohydrin intermediate that is spontaneously rapidly decomposed into the highly fluorescent aldehyde-containing two-photon fluorophore. The probe is shown to exhibit high sensitivity, high specificity, and fast response for HDAC detection in vitro. Imaging studies reveal that the probe is able to directly visualize and monitor HDAC activity in living cells. Moreover, the probe is demonstrated to have the capability of two-photon imaging of HDAC activity in deep tissue slices up to 130 µm. This activatable fluorescent probe affords a useful tool for evaluating HDAC activity and screening HDAC-targeting drugs in both live cell and tissue assays.

A ratiometric fluorescent pH probe based on keto-enol tautomerization for imaging of living cells in extreme acidity.

6-(Diethylamino)-2,3-dihydro-1H-xanthene-4-carbaldehyde (DDXC), a reported synthetic intermediate for near-infrared fluorescent dyes, was developed into a fluorescent pH probe for extreme acidity. The unique sensing mechanism of DDXC for pH is based on the reversible protonation of the carbonyl oxygen followed by keto-enol tautomerization. The probe displays a linear ratiometric fluorescence response (I512/I580) to H+ over the extremely acidic range of pH 2.0-4.0 with a pKa of 3.11, and features high fluorescence quantum yield (Φ = 0.60) and excellent selectivity. More importantly, the probe can be applied to ratiometric fluorescence imaging of pH changes in living cells, making it a potential molecular tool for pH-related cell biology study.