25 research outputs found
Visualizing and Quantifying Protein PolySUMOylation at the Single-Molecule Level
Protein polySUMOylation, the attachment
of small ubiquitin-like
modifier (SUMO) chains to the target protein, is associated with a
variety of physiological processes. However, the analysis of protein
polySUMOylation is often complicated by the heterogeneity of SUMO–target
conjugates. Here, we develop a new strategy to visualize and quantify
polySUMOylation at the single-molecule level by integrating the tetracysteine
(TC) tag labeling technology and total internal reflection fluorescence
(TIRF)-based single-molecule imaging. As a proof-of-concept, we employ
the human SUMO-2 as the model. The addition of TC tag to SUMO-2 can
specifically translate the SUMO-mediated modification into visible
fluorescence signal without disturbing the function of SUMO-2. The
SUMO monomers display homogeneous fluorescence spots at the single-molecule
level, whereas the mixed SUMO chains exhibit nonuniform fluorescence
spots with a wide range of intensities. Analysis of the number and
the brightness of fluorescence spots enable quantitative measurement
of the polySUMOylation degree inside the cells under different physiological
conditions. Due to the frequent occurrence of posttranslational modification
by polymeric chains in cells, this single-molecule strategy has the
potential to be broadly applied for studying protein posttranslational
modification in normal cellular physiology and disease etiology
Highly Sensitive Detection of Protein with Aptamer-Based Target-Triggering Two-Stage Amplification
Highly sensitive detection of proteins is essential to
biomedical
research as well as clinical diagnosis. However, so far most detection
methods rely on antibody-based assays and are usually laborious and
time-consuming with poor sensitivity. Here, we develop a simple and
sensitive method for the detection of a biomarker protein, platelet-derived
growth factor BB (PDGF-BB), based on aptamer-based target-triggering
two-stage amplification. With the involvement of an aptamer-based
probe and an exponential amplification reaction (EXPAR) template,
our method combines strand displacement amplification (SDA) and EXPAR,
transforming the probe conformational change induced by target binding
into two-stage amplification and distinct fluorescence signal. This
detection method exhibits excellent specificity and high sensitivity
with a detection limit of 9.04 × 10<sup>–13</sup> M and
a detection range of more than 5 orders of magnitude, which is comparable
with or even superior to most currently used approaches for PDGF-BB
detection. Moreover, this detection method has significant advantages
of isothermal conditions required, simple and rapid without multiple
separation and washing steps, low-cost without the need of any labeled
DNA probes. Furthermore, this method might be extended to sensitive
detection of a variety of biomolecules whose aptamers undergo similar
conformational changes
Analysis of MicroRNA-Induced Silencing Complex-Involved MicroRNA-Target Recognition by Single-Molecule Fluorescence Resonance Energy Transfer
MicroRNAs (miRNAs) are important regulators of gene expression
that control almost every physiological and pathological process.
Although the complementarity between the seed region of a miRNA and
its target mRNA is usually deemed as the key determinant in the miRNA-target
recognition in animals, the mechanism of their recognition still remains
enigmatic as more and more exceptions challenge the seed rule. Herein,
we employ single-molecule fluorescence resonance energy transfer (smFRET)
to investigate human miRNA-induced silencing complex (miRISC)-involved
miRNA-target recognition with either perfect base pairing or poor
seed match in real time. Our results demonstrate that the recognition
between mammalian miRNA and its target with perfect base pairing proceeds
in a two-state model as prokaryotic guide DNA-mediated recognition,
suggesting a conserved pattern of guide RNA/DNA strand recognition.
In addition to the general rule of miRNA-target recognition, our results
reveal that annealing between miRNA and its target with poor seed
match proceeds in a stepwise way, which is in accordance with the
increase in the number of conformational states of miRNA-target duplex
accommodated by the miRISC, suggesting the structural plasticity of
human miRISC to conciliate the mismatches in seed region. This new
dynamic information revealed by smFRET has an important implication
for comprehensive understanding of the role of miRISC in the target
recognition in mammals
Simple and Accurate Quantification of Quantum Yield at the Single-Molecule/Particle Level
Quantum yield represents one of most fundamental and
important
properties of photoluminescent materials and eventually determines
the suitability of materials for the applications in optical devices,
analysis, biosensing, and fluorescence imaging. Despite that a variety
of methods have been developed to measure the quantum yield, its accurate
quantification still remains a great challenge. Here, we develop a
new approach with the capability to quantify the quantum yield at
the single-molecule/particle level. The quantum yield obtained by
the single-molecule/particle detection is in agreement with that obtained
by the conventional optical method. Importantly, this method can accurately
measure not only the quantum yield of organic dyes but also that of
quantum dots and can be further extended to measure the quantum yield
of various fluorescent materials, including the nontransparent sample
whose quantum yield is impossible to obtain using the conventional
optical method
Sensitive Detection of MicroRNAs with Hairpin Probe-Based Circular Exponential Amplification Assay
MicroRNAs (miRNAs) play important regulatory roles in
a wide range
of biological processes, and their aberrant expression is associated
with cancer development and a variety of diseases. Here, we develop
a simple, sensitive, and specific miRNA assay on the basis of circular
exponential amplification in combination with the hairpin probes.
The binding of target miRNA with a linear DNA template initiates the
first strand displacement amplification (SDA) and generates the universal
triggers which are complementary to the 3′ protruding end of
a hairpin probe. These universal triggers function not only as the
primers to unfold the hairpin probes through an extension reaction,
generating distinct fluorescence signals, but also as the amplification
templates to initiate the second SDA reaction. Moreover, the second
SDA reaction can release new triggers to initiate the above two consecutive
SDA reactions, thus constituting a circular exponential amplification
which enables the conversion of a small amount of miRNAs to a large
number of universal triggers to unfold abundant hairpin probes. This
hairpin probe-based circular exponential amplification assay exhibits
high sensitivity with a detection limit of 3.80 × 10<sup>–13</sup> M and a detection range of 4 orders of magnitude. It can even discriminate
single-nucleotide difference between miRNA family members and perform
well in real sample analysis. Notably, in this assay, the long-stem
hairpin probes are unfolded through an extension reaction rather than
through a conventional hybridization reaction controlled by the thermodynamic
equilibrium in the case of molecular beacons, making the design of
hairpin probes very simple. This hairpin probe-based circular exponential
amplification assay holds a great promise for further application
in biomedical research and early clinical diagnosis
Toward Biocompatible Semiconductor Quantum Dots: From Biosynthesis and Bioconjugation to Biomedical Application
Toward Biocompatible
Semiconductor Quantum Dots: From
Biosynthesis and Bioconjugation to Biomedical Applicatio
Ultrasensitive Detection of Transcription Factors Using Transcription-Mediated Isothermally Exponential Amplification-Induced Chemiluminescence
Transcription factors (TFs) are important
cellular components that
modulate gene expression, and the malregulation of transcription will
lead to a variety of diseases such as cancer and developmental syndromes.
However, the conventional methods for transcription factor assay are
generally cumbersome and costly with low sensitivity. Here, we develop
a label-free strategy for ultrasensitive detection of transcription
factors using a cascade signal amplification of RNA transcription,
dual isothermally exponential amplification reaction (EXPAR), and
G-quadruplex DNAzyme-driven chemiluminescence. Briefly, the specific
binding of TF with the detecting probe prevents the cleavage of the
detecting probe by exonuclease and subsequently facilitates the conversion
of TF signal to abundant RNA triggers in the presence of T7 RNA polymerase.
The obtained RNA triggers can initiate the strand displacement amplification
to yield abundant DNAzymes and DNA triggers, and the released DNA
triggers can further initiate the next rounds of EXPAR reaction. The
synergistic operation of dual EXPAR reaction can produce large amounts
of DNAzymes, which subsequently catalyze the oxidation of luminol
by H<sub>2</sub>O<sub>2</sub> to yield an enhanced chemiluminescence
signal with the assistance of cofactor hemin. Conversely, in the absence
of target TF, the naked detecting probes will be completely digested
by exonucleases, leading to neither the transcription-mediated EXPAR
nor the DNAzyme-driven chemiluminescence signal. This method has a
low detection limit of as low as 6.03 × 10<sup>–15</sup> M and a broad dynamic range from 10 fM to 1 nM and can even measure
the NF-κB p50 of crude cell nuclear extracts. Moreover, this
method can be used to measure a variety of DNA-binding proteins by
simply substituting the target-specific binding sequence in the detecting
probes
Sensitive Detection of Transcription Factors by Isothermal Exponential Amplification-Based Colorimetric Assay
Transcription factors regulate gene expression by binding
to specific
DNA sequences within the regulatory regions of genes and have become
potential targets in clinical diagnosis and drug development. However,
traditional approaches for the detection of transcription factors
are usually laborious and time-consuming with a low sensitivity. Here,
we develop an isothermal exponential amplification reaction (EXPAR)-based
colorimetric assay for simple and sensitive detection of transcription
factor NF-κB p50. In this assay, the presence of NF-κB
p50 is converted to the reporter oligonucleotides through protein–DNA
interaction, exonuclease III digestion, and isothermal exponential
amplification. The subsequent sandwich hybridization of the reporter
oligonucleotides with the gold nanoparticle (AuNP)-labeled DNA probes
generates a red-to-purple color change, allowing the visual detection
of NF-κB p50 with the naked eye. Notably, this method converts
the detection of transcription factors to the detection of DNA without
the requirement of DNA marker-linked antibodies in the case of immuno-PCR
and can sensitively measure NF-κB p50 with a detection limit
of 3.8 pM, which has improved by as much as 4 orders of magnitude
as compared with the conventional AuNP-based colorimetric assay and
the label-free luminescence assay and up to 4 orders of magnitude
as compared with fluorescence resonance energy transfer (FRET)-based
assay as well. Importantly, this method can be used to measure TNF-α-induced
endogenous NF-κB p50 in HeLa cell nuclear extracts and might
be further applied for the detection of various DNA-binding proteins
and aptamer-binding molecules
Phosphorylation-Directed Assembly of a Single Quantum Dot Based Nanosensor for Protein Kinase Assay
Protein
kinases play crucial roles in intracellular signal transduction
and metabolic pathways, and the monitoring of protein kinase activity
is essential to the understanding of fundamental biochemical processes
and the clinical diagnosis. Here, we demonstrate the phosphorylation-directed
assembly of a single quantum dot (QD)-based nanosensor for sensitive
detection of cAMP-dependent protein kinase (PKA). This assay involves
(1) the PKA-directed simultaneous phosphorylation and biotinylation
of cyanine 5 (Cy5)-labeled substrate peptides, (2) the assembly of
phosphorylated and biotinylated peptides onto the surface of the QD,
and (3) the illumination of Cy5 by means of fluorescence resonance
energy transfer (FRET) between the QD and Cy5. With an adenosine triphosphate
(ATP) analogue, γ-biotin-ATP, as the phosphoryl donor, the PKA-catalyzed
phosphorylation reaction incorporates the biotin-conjugated phosphate
group into the substrate peptides to form the biotinylated peptides.
The biotin entity subsequently drives the assembly of peptides onto
the surface of streptavidin-functionalized QD to form the sandwiched
Cy5–peptide–QD nanostructure, enabling the occurrence
of FRET between the QD and Cy5. The FRET signal can be easily recorded
by either the conventional fluorescence spectrometer or the total
internal reflection fluorescence (TIRF) microscope. In contrast, the
absence of PKA cannot lead to the formation of Cy5–peptide–QD
complex and no Cy5 signal can be detected. This protein kinase-actuated
FRET assay is straightforward, without the involvement of either washing
or separation steps, and has a significant advantage of high sensitivity
with a detection limit of 9.3 × 10<sup>–6</sup> U/μL.
Moreover, this method can be used to estimate the half-maximal inhibitory
concentration (IC<sub>50</sub>) value of PKA inhibitor H-89 (<i>N</i>-[2-(<i>p</i>-bromocinnamylamino)Âethyl]-5-isoquinolinesulfonamide
dihydrochloride) and to monitor forskolin (Fsk)/3-isobutyl-1-methylxanthine
(IBMX)-triggered activation of PKA in cell lysates, thus holding great
potential for further applications in protein kinase-related biological
researches and drug discovery
Isothermally Sensitive Detection of Serum Circulating miRNAs for Lung Cancer Diagnosis
Tumor-derived
miRNAs in serum are emerging as the new noninvasive
biomarkers for the diagnosis of human cancers, especially at their
early stage. An ideal method with high sensitivity, excellent selectivity,
a simple procedure, and small amounts of starting materials is imperative
for the detection of clinic circulating miRNAs. Here, we develop a
new method for isothermally sensitive detection of serum miRNAs using
hairpin probe-based rolling circle amplification (HP-RCA). This method
exhibits ultrahigh sensitivity toward lung cancer-related miR-486-5p
with a detection limit of as low as 10 fM and a large dynamic range
of 6 orders of magnitude, and it can even discriminate miR-486-5p
from both miRNAs with high sequence homology and its precursors (pre-miRNAs).
More importantly, this method can directly and accurately distinguish
the expression of serum miR-486-5p among six nonsmall-cell lung carcinoma
(NSCLC) patients and six healthy persons, holding a great potential
for further applications in the clinical diagnosis of lung cancers