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The performance of Stimulated Emission Depletion (STED) microscopy depends critically on the fluorescent probe. Ultrasmall Au nanoclusters (Au NCs) exhibit large Stokes shift, and good stimulated emission response, which are potentially useful for STED imaging. However, Au NCs are polydispersed in size, sensitive to the surrounding environment, and difficult to control surface functional group stoichiometry, which results in reduced density and high heterogeneity in the labeling of biological structures. Here, this limitation is overcome by developing a method to encapsulate ultrasmall Au NCs with DNA cages, which yielded monodispersed, and monofunctionalized Au NCs that are long-term stable. Moreover, the DNA-caging also greatly improved the fluorescence quantum yield and photostability of Au NCs. In STED imaging, the DNA-caged Au NCs yielded ≈40 nm spatial resolution and are able to resolve microtubule line shapes with good labeling density and homogeneity. In contrast, without caging, the Au NCs-DNA conjugates only achieved ≈55 nm resolution and yielded spotted, poorly resolved microtubule structures, due to the presence of aggregates. Overall, a method is developed to achieve precise surface functionalization and greatly improve the monodispersity, stability, as well as optical properties of Au NCs, providing a promising class of fluorescent probes for STED imaging., (© 2024 Wiley‐VCH GmbH.)

A novel fluorescence aptasensor based on DNA charge transport for sensitive protein detection has been developed. A 15nt DNA aptamer against thrombin was used as a model system. The aptamer was integrated into a double strand DNA (dsDNA) that was labeled with a hole injector, naphthalimide (NI), and a fluorophore, Alexa532, at its two ends. After irradiation by UV light, the fluorescence of Alexa532 was bleached due to the oxidization of Alexa532 by the positive charge transported from naphthalimide through the dsDNA. In the presence of thrombin, the binding of thrombin to the aptamer resulted in the unwinding of the dsDNA into ssDNA, which led to the blocking of charge transfer and the strong fluorescence emission of Alexa532. By monitoring the fluorescence signal change, we were able to detect thrombin in homogeneous solutions with high selectivity and high sensitivity down to 1.2 pM. Moreover, as DNA charge transfer is resistant to interferences from biological contexts, the aptasensor can be used directly in undiluted serum with similar sensitivity as that in buffer. This new sensing strategy is expected to promote the exploitation of aptamer-based biosensors for protein assays in complex biological matrixes.

A water-soluble covalently linked C(60)-porphyrin compound-C(60)Por is reported to show enhanced DNA-cleaving activity. The highly enhanced DNA-cleaving activity is expected to be ascribed to the high water-solubility and affinity of C(60)Por to DNA, as well as to the accessibility to the charge-separated states C(60)˙(-)-Por˙(+) formed under radiation.

Molecular beacons (MBs) are specifically designed DNA hairpin structures that are widely used as fluorescent probes. Applications of MBs range from genetic screening, biosensor development, biochip construction, and the detection of single-nucleotide polymorphisms to mRNA monitoring in living cells. The inherent signal-transduction mechanism of MBs enables the analysis of target oligonucleotides without the separation of unbound probes. The MB stem-loop structure holds the fluorescence-donor and fluorescence-acceptor moieties in close proximity to one another, which results in resonant energy transfer. A spontaneous conformation change occurs upon hybridization to separate the two moieties and restore the fluorescence of the donor. Recent research has focused on the improvement of probe composition, intracellular gene quantitation, protein-DNA interaction studies, and protein recognition.

Smad7 is an antagonist of TGF-beta signaling pathway and the mechanism of its inhibitory effect is of great interest. We recently found that Smad7 could function in the nucleus by binding to the DNA elements containing the minimal Smad binding element CAGA box. In this work, we further applied single-molecule force spectroscopy to study the DNA-binding property of Smad7. Smad7 showed similar binding strength to the oligonucleotides corresponding to the CAGA-containing activin responsive element (ARE) and the PAI-1 promoter, as that of Smad4. However, Smad7 also exhibited a binding activity to the mutant ARE with the CAGA sequence substituted, indicating its DNA-binding specificity is different from other Smads. Moreover, we demonstrated that the MH2 domain of Smad7 had a higher binding affinity to the DNA elements than the full-length Smad7, while the N-terminal domain exhibited an inhibitory effect.

The strongly induced circular dichroism (ICD) signals of DNA wrapped around single-walled carbon nanotubes (SWCNTs) are shown by the Synchrotron Radiation Facility. In solution, trace amounts of Hg ions have a strong affinity to bind the nucleic bases of DNA-SWCNTs via a pseudo-first-order kinetic reaction. The Hg binding to the bases of DNA results in partial DNA disassociation from the SWCNTs. Such disassociation of DNA from the SWCNTs will decrease the coupling effects of the transition dipole moments between DNA and SWCNTs, thus inducing the ICD signal of DNA-SWCNTs to decrease significantly. Herein, the ICD of DNA-SWCNTs is applied to detect the concentration of Hg ions at nM level.

We have applied fluorescence anisotropy and fluorescence resonance energy transfer (FRET) techniques to study the interaction between EcoRI DNA methyltransferase (M.EcoRI) and its target DNA in solution. Upon binding with M.EcoRI, the dsDNA containing GAATTC bends to flip out the second adenine for methylation. The binding affinity of M.EcoRI to two dsDNA fragments (20 and 38 bp) was studied with fluorescence anisotropy. Their binding constants at different temperatures from 20 to 40 degrees C were obtained, and the thermodynamic parameters of binding were derived. The results showed that M.EcoRI had a higher binding affinity to the short dsDNA strand than to the long one, and its binding to DNA was primarily entropy-driven. By labeling the 5' ends of the 20-bp dsDNA with two fluorescent dyes, fluorescein (FAM) and tetramethylrhodamine (TMR), we were able to monitor the enhanced TMR fluorescence in the presence of M.EcoRI. The end-to-end distance of the dsDNA determined from the FRET efficiency was changed from 72.4 to 63.4 A, and the DNA bending angle was estimated as 57.8 degrees .

Real-time protein detection in homogeneous solutions is necessary in many biotechnology and biomedical studies. The recent development of molecular aptamers, combined with fluorescence techniques, may provide an easy and efficient approach to protein elucidation. This report describes the development of a fluorescence-based assay with synthetic DNA aptamers that can detect and distinguish molecular variants of proteins in biological samples in a high-throughput process. We used an aptamer with high affinity for the B chain of platelet-derived growth factor (PDGF), labeled it with a fluorophore and a quencher at the two termini, and measured fluorescence quenching by PDGF. The specific quenching can be used to detect PDGF at picomolar concentrations even in the presence of serum and other cell-derived proteins in cell culture media. This is the first successful application of a synthetic aptamer for the detection of tumor-related proteins directly from the tumor cells. We also show that three highly related molecular variants of PDGF (AA, AB, and BB dimers) can be distinguished from one another in this single-step assay, which can be readily adapted to a microtiter plate assay for high-throughput analysis. The use of fluorescence quenching as a measure of binding between the DNA probe and the target protein eliminates potential false signals that may arise in traditional fluorescence enhancement assays as a result of degradation of the DNA aptamer by contaminating nucleases in biological specimens. This assay is applicable to proteins that are not naturally DNA binding. The excellent specificity, ultrahigh sensitivity, and simplicity of this one-step assay addresses a growing need for high-throughput methods that detect changes in the expression of gene products and their variants in cell cultures and biological specimens.

NS2 protein is essential for hepatitis C virus (HCV) replication. NS2 protein was expressed and purified. Aptamers against NS2 protein were raised and antiviral effects of the aptamers were examined. The molecular mechanism through which the aptamers exert their anti-HCV activity was investigated. The data showed that aptamer NS2-3 inhibited HCV RNA replication in replicon cell line and infectious HCV cell culture system. NS2-3 and another aptamer NS2-2 were demonstrated to inhibit infectious virus production without cytotoxicity in vitro. They did not affect hepatitis B virus replication. Interferon beta (IFN-β) and interferon-stimulated genes (ISGs) were not induced by the aptamers in HCV-infected hepatocytes. Furthermore, our study showed that N-terminal region of NS2 protein is involved in the inhibition of HCV infection by NS2-2. I861T within NS2 is the major resistance mutation identified. Aptamer NS2-2 disrupts the interaction of NS2 with NS5A protein. The data suggest that NS2-2 aptamer against NS2 protein exerts its antiviral effects through binding to the N-terminal of NS2 and disrupting the interaction of NS2 with NS5A protein. NS2-specific aptamer is the first NS2 inhibitor and can be used to understand the mechanisms of virus replication and assembly. It may be served as attractive candidates for inclusion in the future HCV direct-acting antiviral combination therapies. [ABSTRACT FROM AUTHOR]

DNA nanotrain: Anchoring of preformed fluorescent DNA nanodevices (NDs; see picture) and in situ self‐assembly of fluorescent DNA NDs on target living cell surfaces are reported. The in situ self‐assembly of the nanodevice was further shown on surfaces of living cells in cell mixtures. These DNA NDs exhibited fluorescence emission and underwent fluorescence resonance energy transfer (FRET) on living cell surfaces. [ABSTRACT FROM AUTHOR]

Atomic force microscopy (AFM) was employed to quantitatively detect the interaction between a transcription factor, ZmDREB1A, and its target DNA sequence (DRE). The specificity of the interaction was revealed by comparing the binding probability of ZmDREB1A to DRE element with core sequence ACCGAC and to the ERE element with core sequence AGCCGCC. Moreover, the single-molecular unbinding force between ZmDREB1A and DRE element was determined using Poisson statistical method to be 99±9 pN. The results indicated that AFM could serve as a sensitive and reliable technique to probe the interaction properties of the transcription factor and DNA element at single-molecular level, thus indicating that AFM is advantageous over the traditional methods, such as the electrophoretic mobility shift assay, regarding to the functional analysis of transcription factors. [ABSTRACT FROM AUTHOR]

Nucleic acid aptamers are single-strandedRNA or DNA molecules that bind to their targets with high specificityand affinity. Although their biomedical applications have been booming,it is still debatable whether an aptamer recognizes its target through“induced fit” or “conformational selection”,a central question in molecular recognition. To address this question,an ATP-binding DNA aptamer was selected as a model system and theconformational properties of this aptamer with and without the presenceof ATP were investigated by single-pair Förster resonance energytransfer (spFRET) spectroscopy. The single-molecule results indicatethat the aptamer can fold into a double-stranded-like structure, similarto the ligand-bound conformation, even without the presence of ATP.The folded structure is thermally stable at high salt concentrationsand becomes rather dynamic at low salt concentrations. Although inthe latter condition, the aptamer prefers unfolded structures, itcan occasionally migrate to the folded conformation for a short timebefore being unfolded again. The binding of ATP to the aptamer stabilizesthe folded structure, which populates the ligand-bound state of theaptamer, thus shifting the conformational equilibrium. Collectively,our data support that the ATP-binding DNA aptamer recognizes ATP ligandthrough “conformational selection”. [ABSTRACT FROM AUTHOR]

Abstract: The interaction force between transcription factor TINY and its corresponding cis-element has been studied with single molecule force microscopy. TINY belongs to the AP2/EREBP family, which is the second largest transcription factor family in plant and plays an important role in the genetic regulation of plant development and growth. The results showed that TINY bound to dehydration-responsive element (DRE) with the core sequence A/GCCGAC efficiently. The single molecule forces between TINY and DRE A/GCCGAC measured by atomic force microscopy were 83.5±3.4 and 81.4±4.9pN, respectively. Either the single base substitution of the DRE core sequence or point mutation of the key amino acid in TINY DNA-binding domain greatly reduced the binding strength. We have previously reported the method of using AFM to measure the interaction force between transcription factor ZmDREB1A, another AP2/EREBP family protein, and DRE responsive element. The results in this work further confirm the applicability and high sensitivity of AFM measurements in transcription factor–DNA interaction study. More interesting, it was found that 19th amino acid mutation of TINY resulted in the decrease of the binding force but the binding probability was the same as the wild type TINY. The reduced binding force was correlated with the reduced activity of TINY regulated reporter gene in the yeast one-hybrid experiment. It is thus expected that the single molecule force measurement offers valuable information on the analysis of transcription factor regulated gene expression. [Copyright &y& Elsevier]