Search

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]