Facile Synthesis of Aptamer-Functionalized Polydopamine-Coated Magnetic Graphene Oxide Nanocomposites for Highly Efficient Purification of His-Tagged Proteins.

Recombinant proteins hold significant importance in numerous disciplines. As the demand for expressing and purifying these proteins grows, the scientific community is in dire need of a simple yet versatile methodology that can efficiently purify these proteins. Aptamers as synthetic nucleic acid-based ligands with high affinity have shown promise in this regard, as they can capture targets through molecular recognition. In this study, novel aptamer-functionalized polydopamine-coated magnetic graphene oxide nanocomposites were facilely prepared, achieving an impressive average aptamer coverage density (45 nmol/mg). These nanocomposites exhibited a uniform structure and robust magnetic responsiveness. The findings indicated that they possess several advantages, such as rapid adsorption, substantial capacity (171.4 mg/g), and excellent reusability. Notably, due to the inherent properties of nucleic acids, the immobilized aptamer-magnetic beads can be utilized repeatedly with high purification efficiency. Finally, the nanocomposites were further employed to purify His-tagged proteins from actual samples. Remarkably, they were able to selectively and efficiently isolate His-tagged retinoid X receptor alpha protein from complex Escherichia coli lysate. The purified His-tagged retinoid X receptor alpha protein was analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This confirmed the efficacy of developed nanocomposites, reinforcing their vast potential for purification of His-tagged recombinant proteins.

Functional DNA Superstructures Exhibit Positive Homotropic Allostery in Ligand Binding.

Inspired by intrinsically disordered proteins in nature, DNA aptamers can be engineered to display strongly homotropic allosteric (or cooperative) ligand binding, representing a unique feature that could be of great utility in applications such as biosensing, imaging and drug delivery. The use of an intrinsic disorder mechanism, however, comes with an inherent drawback of significantly reduced overall binding affinity. We hypothesize that it could be addressed via the design of multivalent supramolecular aptamers. We built functional DNA superstructures (denoted as 3D DNA), made of long-chain DNA containing tandem repeating DNA aptamers (or concatemeric aptamers). The 3D DNA systems exhibit highly cooperative binding to both small molecules and proteins, without loss of binding affinities of their parent aptamers. We further produced a highly responsive sensor for fluorescence imaging of glutamate stimulation-evoked adenosine triphosphate (ATP) release in neurons, as well as force stimulus-triggered ATP release in astrocytes.

In Vitro Selection of Circular DNA Aptamers for Biosensing Applications.

We report on the first effort to select DNA aptamers from a circular DNA library, which resulted in the discovery of two high-affinity circular DNA aptamers that recognize the glutamate dehydrogenase (GDH) from Clostridium difficile, an established antigen for diagnosing Clostridium difficile infection (CDI). One aptamer binds effectively in both the circular and linear forms, the other is functional only in the circular configuration. Interestingly, these two aptamers recognize different epitopes on GDH, demonstrating the advantage of selecting aptamers from circular DNA libraries. A sensitive diagnostic test was developed to take advantage of the high stability of circular DNA aptamers in biological samples and their compatibility with rolling circle amplification. This test is capable of identifying patients with active CDI using stool samples. This work represents a significant step forward towards demonstrating the practical utility of DNA aptamers in clinical diagnosis.

DNAzyme Feedback Amplification: Relaying Molecular Recognition to Exponential DNA Amplification.

Technologies capable of linking DNA amplification to molecular recognition are very desirable for ultrasensitive biosensing applications. We have developed a simple but powerful isothermal DNA amplification method, termed DNAzyme feedback amplification (DFA), that is capable of relaying molecular recognition to exponential DNA amplification. The method incorporates both an RNA-cleaving DNAzyme (RCD) and rolling circle amplification (RCA) carried out by a special DNA polymerase using a circular DNA template. DFA begins with a stimulus-dependent RCA reaction, producing tandemly linked RCDs in long-chain DNA products. These RCDs cleave an RNA-containing DNA sequence to form additional primers that hybridize to the circular DNA molecule, giving rise to DNA assemblies that act as the new inputs for RCA. The RCA reaction and the cleavage event keep on feeding each other autonomously, resulting in exponential growth of repetitive DNA sequences that can be easily detected. This method can be used for the detection of both nucleic acid based targets and non-nucleic acid analytes. In this article, we discuss the conceptual framework of the feedback amplification approach, the essential features of this method as well as remaining challenges and possible solutions.

Selection and characterization of DNA aptamers for detection of glutamate dehydrogenase from Clostridium difficile.

Rapid and accurate diagnosis of Clostridium difficile infections (CDI) is crucial for patient treatment, infection control and epidemiological monitoring. As an important antigen, glutamate dehydrogenase (GDH) has been proposed as a preliminary screening test target for CDI. However, current assays based on GDH activity or GDH immunoassays have suboptimal sensitivity and specificity. Herein, we describe the selection and characterization of single-stranded DNA aptamers that specifically target GDH. After 10 rounds of selection, high-throughput sequencing was used to identify enriched aptamer candidates. Of 10 candidates, three aptamers for GDH were identified. Gel shift assays showed that these aptamers exhibited low nanomolar affinities. One aptamer was optimized based on structural analysis and further engineered into a structure-switching fluorescence signaling aptamer, wherein desorption from reduced graphene oxide (RGO) upon binding of GDH led to an increase in fluorescence emission. This method allowed for quantitative detection of GDH with a detection limit of 1 nM, providing great potential for its further application in CDI diagnosis.

A novel environmental fate of graphene oxide: Biodegradation by a bacterium Labrys sp. WJW to support growth.

Graphene oxide (GO) is a new type of nanomaterial with unique physicochemical properties and diverse applications, whereas it poses potential risk to human and environment. By screening from natural soil exposed to GO in the laboratory, we successfully obtained a novel bacterium, Labrys sp. WJW, which was able to use GO as the sole carbon source for growth. Within 8 days, cell numbers increased 16.76 ±â€¯3.21 folds using 100 mg/L GO as the carbon source by qPCR analysis. The bacterial biodegradation which resulted in formation of holes and functional group changes of GO was proved by Raman spectroscopy, atomic force microscopy, transmission electron microscopy and X-ray photoelectron spectroscopy analyses. Aromatic intermediates with structures of benzoic acid and phenol were identified using gas chromatograph-mass spectrometry and liquid chromatography/time-of-flight/mass spectrometry. Combination of genomic and proteomic analyses were performed to explore the proteins associated with GO degradation. A total of 644 proteins were significantly shifted. Bioinformatics analysis indicated that part of the up-regulated proteins were related to oxidation, ring cleavage and intermediates transmembrane processes, and GO was supposed to be degraded to benzoate and further degraded for downstream processes. This study enriches our understanding and provides new insights into the environmental fate of GO.

Comparison of gold nanoparticles biosynthesized by cell-free extracts of Labrys, Trichosporon montevideense, and Aspergillus.

Biosynthesis of gold nanoparticles (AuNPs) by microbes has received much attention as an efficient and eco-friendly process. However, the characteristics of AuNPs biosynthesized by different microbial cell-free extracts are rarely comparatively studied. In this study, three locally isolated strains, i.e., bacteria Labrys sp. WJW, yeast Trichosporon montevideense WIN, and filamentous fungus Aspergillus sp. WL-Au, were selected for AuNPs biosynthesis. UV-Vis absorption bands at 538, 539, and 543 nm confirmed the formation of AuNPs by these strains. Transmission electron microscopy and selected area electron diffraction analyses revealed that the as-synthesized AuNPs were crystalline with spherical or pseudo-spherical shapes. However, the average sizes of these AuNPs were diverse, which were 18.8, 22.2 and 9.5 nm, respectively. The biomolecules involved in nanoparticles stabilization were demonstrated by Fourier transform infrared spectroscopy analysis. Four common functional groups such as -N-H, -C=C, -N=O, and -S=O groups were detected in these AuNPs, while a distinct -C=O group was involved in WL-Au-AuNPs. The catalytic rate of WL-Au-AuNPs toward 4-nitrophenol reduction (0.37 min-1) was much higher than those of others (WJW-AuNPs 0.27 min-1 and WIN-AuNPs 0.23 min-1). This research would provide useful information for exploring efficient microbial candidates to synthesize AuNPs with excellent performances.

Silenced suppressor of cytokine signaling 1 (SOCS1) enhances the maturation and antifungal immunity of dendritic cells in response to Candida albicans in vitro.

Dendritic cells (DCs) are known to play an important role in initiating and orchestrating antimicrobial immunity. Given the fact that candidiasis appears often in immunocompromised patients, it seems plausible that DCs hold the key to new antifungal strategies. One possibility to enhance the potency of DC-based immunotherapy is to silence the negative immunoregulatory pathways through the ablation suppressor of cytokine signaling suppressor 1 (SOCS1). Here, we deliver small interfering RNA (siRNA) against SOCS1 into murine bone marrow DCs, and as a consequence, we investigate the maturation/action of DCs and the subsequent T cell response after exposure to C. albicans. Our results show that the maturation of DCs (i.e., expressions of CD80, CD40, CD86, and MHC II) are significantly increased in the silenced SOCS1 treatment group after exposure to C. albicans. As a result, suppression of the SOCS1 promotes the greatest expression of IFN-γ and IL-12, and reduces IL-4 secretions, which induce CD4(+) cell Th1 differentiation but inactivate Th2 cell development. The responses of IL-6 and TNF-ß consist of up-regulation in the presence of C. albicans, but this is not specific to SOCS1 silencing, suggesting that these cytokines are not regulated by the SOCS1 gene in fungal infections. We find Th17 differentiation is unchanged regardless of SOCS1 inhibition. The increase in phagocytosis and killing of C. albicans in SOCS1 gene-treated DCs indicate a role for this cytokine suppressor in innate immunity as well. In conclusion, our findings support the view that SOCS1 protein is a critical inhibitory molecule for controlling cytokine response and antigen presentation by DCs, thereby regulating the magnitude of innate and adaptive immunities by generating IFN-γ-production T cells (Th1)--but not Th17--from naïve CD4(+) T cells. Our study demonstrates that SOCS1 siRNA can serve as a useful vehicle to modulate the function of DCs against C. albicans infection.