Ionic liquid-based aqueous biphasic system as an alternative tool for enhanced bacterial DNA extraction.

BACKGROUND: Developing an alternative and benign method for DNA extraction is imperative due to the high cost and potential harms associated with conventional techniques. Investigation of Ionic liquid (IL) as a solvent for DNA storage and stability revealed the ability of IL to assist DNA processes. IL-based aqueous biphasic system emerges as a comprehensive extraction platform capitalizing on the task-specificity of ILs and the wide applicability of ABS for biomolecule extractions. Therefore, it is beneficial to optimize an IL-based ABS specifically for DNA extraction, taking into account the fundamental interactions between the IL and DNA. RESULTS: The primary objective was to design ABS consisting of Ammonium based ILs, and Potassium phosphate buffer as the salting-out agent for the partitioning of salmon sperm DNA. The analysis focused on optimizing biocompatible anions for the extraction. Moreover, the stability of the DNA in the IL rich phases was analysed to validate the method. The proposed process was then employed for extracting plasmid DNA from bacteria, demonstrating results comparable to those obtained with a commercially available kit. Further validation using agarose gel electrophoresis and transformation of the extracted DNA into E.coli were conducted, producing promising outcomes. Although there is room for improvement in terms of recovery of DNA and reusability of ABS, the described approach is comparable with the conventional one while being cost-effective, and showcases a noticeable and convincing link to eco-friendly processes. SIGNIFICANCE: There is limited literature on IL-based ABS for DNA extraction, and the existing studies predominantly concentrate on systems derived from Cholinium ILs. However, their high hydrophilicity limits the choice of the second-phase forming component to polymers for the formation of ABS. Ammonium ILs efficiently form biphasic systems with various available salting-out agents, and biocompatible anions are introduced to mitigate the toxicity of the ILs.

Single-Molecule Optical Tweezers As a Tool for Delineating the Mechanisms of Protein-Processing Mechanoenzymes.

Mechanoenzymes convert chemical energy from the hydrolysis of nucleotide triphosphates to mechanical energy for carrying out cellular functions ranging from DNA unwinding to protein degradation. Protein-processing mechanoenzymes either remodel the protein structures or translocate them across cellular compartments in an energy-dependent manner. Optical-tweezer-based single-molecule force spectroscopy assays have divulged information on details of chemo-mechanical coupling, directed motion, as well as mechanical forces these enzymes are capable of generating. In this review, we introduce the working principles of optical tweezers as a single-molecule force spectroscopy tool and assays developed to decipher the properties such as unfolding kinetics, translocation velocities, and step sizes by protein remodeling mechanoenzymes. We focus on molecular motors involved in protein degradation and disaggregation, i.e., ClpXP, ClpAP, and ClpB, and insights provided by single-molecule assays on kinetics and stepping dynamics during protein unfolding and translocation. Cellular activities such as protein synthesis, folding, and translocation across membranes are also energy dependent, and the recent single-molecule studies decoding the role of mechanical forces on these processes have been discussed.