7 research outputs found
Genetically Encoded Fluorescent RNA Sensor for Ratiometric Imaging of MicroRNA in Living Tumor Cells
Light-up
RNA aptamers are valuable tools for fluorescence imaging of RNA in
living cells and thus for elucidating RNA functions and dynamics.
However, no light-up RNA sensor has been reported for imaging of microRNAs
(miRs) in mammalian cells. We report a novel genetically encoded RNA
sensor for fluorescent imaging of miRs in living tumor cells using
a light-up RNA aptamer that binds to sulforhodamine and separates
it from a conjugated contact quencher. On the basis of the structural
switching mechanism for molecular beacon, we show that the RNA sensor
activates high-contrast fluorescence from the sulforhodamine-quencher
conjugate when its stem–loop responsive motif hybridizes with
target miR. The RNA sensor can be stably expressed within a designed
tRNA scaffold in tumor cells and deliver light-up response to miR
target. We also realize the RNA sensor for dual-emission, ratiometric
imaging by coexpression of RNA sensor with GFP, enabling quantitative
studies of target miR in living cells. Our design may provide a new
paradigm for developing robust, sensitive light-up RNA sensors for
RNA imaging applications
Structure-Switching Aptamer Triggering Hybridization Chain Reaction on the Cell Surface for Activatable Theranostics
The ability to probe low-abundance
biomolecules or transport a
high-load drug in target cells is essential for biology and theranostics.
We develop a novel activatable theranostic approach by using a structure-switching
aptamer triggered hybridization chain reaction (HCR) on the cell surface,
which for the first time creates an aptamer platform enabling real-time
activation and amplification for fluorescence imaging and targeting
therapy. The aptamer probe is designed not to initiate HCR in its
free state but trigger HCR on binding to the target cell via structure
switching. The HCR not only amplifies fluorescence signals from a
fluorescence-quenched probe for activatable tumor imaging but also
accumulates high-load prodrugs from a drug-labeled probe and induces
its uptake and conversion into cisplatin in cells for selective tumor
therapy. An in vitro assay shows that this approach affords efficient
signal amplification for fluorescence detection of target protein
tyrosine kinase-7 (PTK7) with a detection limit of 1 pM. Live cell
studies reveal that it provides high-contrast fluorescence imaging
and highly sensitive detection of tumor cells, while renders high-efficiency
drug delivery into tumor cells via an endocytosis pathway. The results
imply the potential of the developed approach as a promising platform
for early stage diagnosis and precise therapy of tumors
Electrostatic Nucleic Acid Nanoassembly Enables Hybridization Chain Reaction in Living Cells for Ultrasensitive mRNA Imaging
Efficient
approaches for intracellular delivery of nucleic acid
reagents to achieve sensitive detection and regulation of gene and
protein expressions are essential for chemistry and biology. We develop
a novel electrostatic DNA nanoassembly that, for the first time, realizes
hybridization chain reaction (HCR), a target-initiated alternating
hybridization reaction between two hairpin probes, for signal amplification
in living cells. The DNA nanoassembly has a designed structure with
a core gold nanoparticle, a cationic peptide interlayer, and an electrostatically
assembled outer layer of fluorophore-labeled hairpin DNA probes. It
is shown to have high efficiency for cellular delivery of DNA probes
via a unique endocytosis-independent mechanism that confers a significant
advantage of overcoming endosomal entrapment. Moreover, electrostatic
assembly of DNA probes enables target-initialized release of the probes
from the nanoassembly via HCR. This intracellular HCR offers efficient
signal amplification and enables ultrasensitive fluorescence activation
imaging of mRNA expression with a picomolar detection limit. The results
imply that the developed nanoassembly may provide an invaluable platform
in low-abundance biomarker discovery and regulation for cell biology
and theranostics
Activity-Based DNA-Gold Nanoparticle Probe as Colorimetric Biosensor for DNA Methyltransferase/Glycosylase Assay
We
have developed a novel biosensor platform for colorimetric detection
of active DNA methyltransferase/glycosylase based on terminal protection
of the DNA-gold nanoparticle (AuNP) probes by mechanistically covalent
trapping of target enzymes. This biosensor relied on covalent capture
of target enzymes by activity-based DNA probes which created terminal
protection of the DNA probes tethered on AuNPs from degradation by
Exo I and III. This biosensor has the advantages of having highly
sensitive, rapid, and convenient detection due to its use of the homogeneous
assay format and strong surface plasmon absorption. Because the activity-based
probes (ABPs) are mechanistically specific to target enzymes, this
strategy also offers improved selectivity and can achieve the information
about both abundance and activity of the enzymes. We have demonstrated
this strategy using a human DNA (cytosine-5) methyltransferase (Dnmt
1) and a human 8-oxoguanine glycosylase (hOGG 1). The results reveal
that the colorimetric response increases dynamically with increasing
activity of the enzymes, implying a great potential of this strategy
for DNA methyltransferase/glycosylase detection and molecular diagnostics
and drug screening. Our strategy can also be used as a promising and
convenient approach for visualized screening of ABPs for DNA modifying
enzymes
Amphiphilic BODIPY-Based Photoswitchable Fluorescent Polymeric Nanoparticles for Rewritable Patterning and Dual-Color Cell Imaging
Photoswitchable fluorescent polymeric
nanoparticles (PFPNs) with
controllable molecular weight, high contrast, biocompatibility, and
prominent photostability are highly desirable but still scarce for
rewritable printing, super-resolution bioimaging, and rewritable data
storage. In this study, novel amphiphilic BODIPY-based PFPNs with
considerable merits are first synthesized by a facile one-pot RAFT-mediated
miniemulsion polymerization method. The polymerization is performed
by adopting polymerizable BODIPY and spiropyran derivatives, together
with MMA as monomer, and mediated by utilizing biocompatible PEO macro-RAFT
agent as both control agent and reactive stabilizer. The amphiphilic
BODIPY-based PFPNs not only exhibit reversibly photoswitchable fluorescence
properties under the alternative UV and visible light illumination
through induced intraparticle fluorescence resonance energy transfer
(FRET) but also display controllable molecular weight with narrow
polydispersity index (PDI), high contrast of fluorescence, tunable
energy transfer efficiency, good biocompatibility, excellent photostability,
favorable photoreversibility, etc. The as-prepared PFPNs are successfully
demonstrated for rewritable fluorescence patterning and high-contrast
dual-color fluorescence imaging of living cells, implying its potential
for rewritable data storage and broad biological applications in cell
biology and diagnostics
Self-Catalytic Growth of Unmodified Gold Nanoparticles as Conductive Bridges Mediated Gap-Electrical Signal Transduction for DNA Hybridization Detection
A simple and sensitive gap-electrical
biosensor based on self-catalytic
growth of unmodified gold nanoparticles (AuNPs) as conductive bridges
has been developed for amplifying DNA hybridization events. In this
strategy, the signal amplification degree of such conductive bridges
is closely related to the variation of the glucose oxidase (GOx)-like
catalytic activity of AuNPs upon interaction with single- and double-stranded
DNA (ssDNA and dsDNA), respectively. In the presence of target DNA,
the obtained dsDNA product cannot adsorb onto the surface of AuNPs
due to electrostatic interaction, which makes the unmodified AuNPs
exhibit excellent GOx-like catalytic activity. Such catalytic activity
can enlarge the diameters of AuNPs in the glucose and HAuCl<sub>4</sub> solution and result in a connection between most of the AuNPs and
a conductive gold film formation with a dramatically increased conductance.
For the control sample, the catalytic activity sites of AuNPs are
fully blocked by ssDNA due to the noncovalent interaction between
nucleotide bases and AuNPs. Thus, the growth of the assembled AuNPs
will not happen and the conductance between microelectrodes will be
not changed. Under the optimal experimental conditions, the developed
strategy exhibited a sensitive response to target DNA with a high
signal-to-noise ratio. Moreover, this strategy was also demonstrated
to provide excellent differentiation ability for single-nucleotide
polymorphism. Such performances indicated the great potential of this
label-free electrical strategy for clinical diagnostics and genetic
analysis under real biological sample separation
DataSheet_1_Identification and validation of potential diagnostic signature and immune cell infiltration for NAFLD based on cuproptosis-related genes by bioinformatics analysis and machine learning.docx
Background and aimsCuproptosis has been identified as a key player in the development of several diseases. In this study, we investigate the potential role of cuproptosis-related genes in the pathogenesis of nonalcoholic fatty liver disease (NAFLD).MethodThe gene expression profiles of NAFLD were obtained from the Gene Expression Omnibus database. Differential expression of cuproptosis-related genes (CRGs) were determined between NAFLD and normal tissues. Protein–protein interaction, correlation, and function enrichment analyses were performed. Machine learning was used to identify hub genes. Immune infiltration was analyzed in both NAFLD patients and controls. Quantitative real-time PCR was employed to validate the expression of hub genes.ResultsFour datasets containing 115 NAFLD and 106 control samples were included for bioinformatics analysis. Three hub CRGs (NFE2L2, DLD, and POLD1) were identified through the intersection of three machine learning algorithms. The receiver operating characteristic curve was plotted based on these three marker genes, and the area under the curve (AUC) value was 0.704. In the external GSE135251 dataset, the AUC value of the three key genes was as high as 0.970. Further nomogram, decision curve, calibration curve analyses also confirmed the diagnostic predictive efficacy. Gene set enrichment analysis and gene set variation analysis showed these three marker genes involved in multiple pathways that are related to the progression of NAFLD. CIBERSORT and single-sample gene set enrichment analysis indicated that their expression levels in macrophages, mast cells, NK cells, Treg cells, resting dendritic cells, and tumor-infiltrating lymphocytes were higher in NAFLD compared with control liver samples. The ceRNA network demonstrated a complex regulatory relationship between the three hub genes. The mRNA level of these hub genes were further confirmed in a mouse NAFLD liver samples.ConclusionOur study comprehensively demonstrated the relationship between NAFLD and cuproptosis, developed a promising diagnostic model, and provided potential targets for NAFLD treatment and new insights for exploring the mechanism for NAFLD.</p