Genetically Encoded Fluorogenic DNA Aptamers for Imaging Metabolite in Living Cells.

Genetically encoded fluorescent protein and fluorogenic RNA sensors are indispensable tools for imaging biomolecules in cells. To expand the toolboxes and improve the generalizability and stability of this type of sensor, we report herein a genetically encoded fluorogenic DNA aptamer (GEFDA) sensor by linking a fluorogenic DNA aptamer for dimethylindole red with an ATP aptamer. The design enhances red fluorescence by 4-fold at 650 nm in the presence of ATP.

Fluorogenic Aptamer Optimizations on a Massively Parallel Sequencing Platform.

F luorogenic ap tamers (FAPs) have become an increasingly important tool in cellular sensing and pathogen diagnostics. However, fine-tuning FAPs for enhanced performance remains challenging even with the structural details provided by X-ray crystallography. Here we present a novel approach to optimize a DNA-based FAP (D-FAP), Lettuce, on repurposed Illumina next-generation sequencing (NGS) chips.

Two-photon autofluorescence lifetime assay of rabbit photoreceptors and retinal pigment epithelium during light-dark visual cycles in rabbit retina.

Two-photon excited fluorescence (TPEF) is a powerful technique that enables the examination of intrinsic retinal fluorophores involved in cellular metabolism and the visual cycle. Although previous intensity-based TPEF studies in non-human primates have successfully imaged several classes of retinal cells and elucidated aspects of both rod and cone photoreceptor function, fluorescence lifetime imaging (FLIM) of the retinal cells under light-dark visual cycle has yet to be fully exploited. Here we demonstrate a FLIM assay of photoreceptors and retinal pigment epithelium (RPE) that reveals key insights into retinal physiology and adaptation.

A non-FRET DNA reporter that changes fluorescence colour upon nuclease digestion.

Fluorescence resonance energy transfer (FRET) reporters are commonly used in the final stages of nucleic acid amplification tests to indicate the presence of nucleic acid targets, where fluorescence is restored by nucleases that cleave the FRET reporters. However, the need for dual labelling and purification during manufacturing contributes to the high cost of FRET reporters. Here we demonstrate a low-cost silver nanocluster reporter that does not rely on FRET as the on/off switching mechanism, but rather on a cluster transformation process that leads to fluorescence color change upon nuclease digestion.

Multiplexed imaging in live cells using pulsed interleaved excitation spectral FLIM.

Multiplexed fluorescence detection has become increasingly important in the fields of biosensing and bioimaging. Although a variety of excitation/detection optical designs and fluorescence unmixing schemes have been proposed to allow for multiplexed imaging, rapid and reliable differentiation and quantification of multiple fluorescent species at each imaging pixel is still challenging. Here we present a pulsed interleaved excitation spectral fluorescence lifetime microscopic (PIE-sFLIM) system that can simultaneously image six fluorescent tags in live cells in a single hyperspectral snapshot.

Computational study on the binding of Mango-II RNA aptamer and fluorogen using the polarizable force field AMOEBA.

Fluorescent light-up aptamers (FLAPs) are well-performed biosensors for cellular imaging and the detection of different targets of interest, including RNA, non-nucleic acid molecules, metal ions, and so on. They could be easily designed and emit a strong fluorescence signal once bound to specified fluorogens. Recently, one unique aptamer called Mango-II has been discovered to possess a strong affinity and excellent fluorescent properties with fluorogens TO1-Biotin and TO3-Biotin.

Massively Parallel Selection of NanoCluster Beacons.

NanoCluster Beacons (NCBs) are multicolor silver nanocluster probes whose fluorescence can be activated or tuned by a proximal DNA strand called the activator. While a single-nucleotide difference in a pair of activators can lead to drastically different activation outcomes, termed polar opposite twins (POTs), it is difficult to discover new POT-NCBs using the conventional low-throughput characterization approaches. Here, a high-throughput selection method is reported that takes advantage of repurposed next-generation-sequencing chips to screen the activation fluorescence of ≈40 000 activator sequences.

Generative adversarial network enables rapid and robust fluorescence lifetime image analysis in live cells.

Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to quantify molecular compositions and study molecular states in complex cellular environment as the lifetime readings are not biased by fluorophore concentration or excitation power. However, the current methods to generate FLIM images are either computationally intensive or unreliable when the number of photons acquired at each pixel is low. Here we introduce a new deep learning-based method termed flimGANE (fluorescence lifetime imaging based on Generative Adversarial Network Estimation) that can rapidly generate accurate and high-quality FLIM images even in the photon-starved conditions.

Measuring DNA Hybridization Kinetics in Live Cells Using a Time-Resolved 3D Single-Molecule Tracking Method.

Single-molecule detection enables direct characterization of annealing/melting kinetics of nucleic acids without the need for synchronization of molecular states, but the current experiments are not carried out in a native cellular context. Here we describe an integrated 3D single-molecule tracking and lifetime measurement method that can follow individual DNA molecules diffusing inside a mammalian cell and observe multiple annealing and melting events on the same molecules. By comparing the hybridization kinetics of the same DNA strand , we found the association constants can be 13- to 163-fold higher in the molecular crowding cellular environment.

Assessing metastatic potential of breast cancer cells based on EGFR dynamics.

Derailed transmembrane receptor trafficking could be a hallmark of tumorigenesis and increased tumor invasiveness, but receptor dynamics have not been used to differentiate metastatic cancer cells from less invasive ones. Using single-particle tracking techniques, we  developed a phenotyping asssay named Transmembrane Receptor Dynamics (TReD), studied the dynamics of epidermal growth factor receptor (EGFR) in seven breast epithelial cell lines and developed a phenotyping assay named Transmembrane Receptor Dynamics (TReD). Here we show a clear evidence that increased EGFR diffusivity and enlarged EGFR confinement size in the plasma membrane (PM) are correlated with the enhanced metastatic potential in these cell lines.

Improving NanoCluster Beacon performance by blocking the unlabeled NC probes.

While NanoCluster Beacon (NCB) is a versatile molecular probe, it suffers from a low target-specific signal issue due to impurities. Here we show that adding a "blocker" strand to the reaction can effectively block the nonfunctional probes and enhance the target-specific signal by 14 fold at a 0.1 target/probe ratio.