Hafif deterjanda saflaştirilmiş l-arjinin/agmatin antiport membran proteinini hedefleyen yeni 2′f-PY RNA aptamerlerinin sistematik evrimi

Bacterial microorganisms have developed numerous and diverse systems to handle potentially detrimental acidic conditions in their external environment. In particular, some pathogenic and other nonpathogenic enteric bacteria have a number of exceptional and well-organized acid resistance (AR) mechanisms that work together to counter intracellular acidification and damage and enable survival under the extreme acidic conditions of the mammalian stomach. The common Escherichia coli with both its virulent and benign strains is particularly remarkable considering its adaptation to neutral pH. In these bacteria, the challenge of a low external pH is efficiently mitigated by highly potent proton consumption AR systems that consist of two generic components: a cytoplasmic pyrodoxal-5′-phoshpate (PLP)-dependent amino acid decarboxylase and an inner plasma membrane amino acid antiporter. Both, decarboxylation of a substrate amino acid into a product and CO2 in a proton-dependent PLP-catalyzed manner and antiporter-mediated exchange of the internally generated decarboxylation products with the externally located substrate amino acid maintain the system running and aid in restoring pH homeostasis of the cell. One of these systems is the arginine-dependent acid resistance (ADAR) system constituted by acid-induced arginine decarboxylase (AdiA) and arginine-agmatine antiporter (AdiC). The impressive competence of AR systems and especially those used by infectious, disease-causing enteric bacteria, comes at a big cost—the well-being and health of humans. Henceforth, in this work, for the first time, we raised five candidate 2′FY RNA candidate aptamers (noorA, noorB, noorC, AdiC19 and AdiC72) against the ADAR system's integral membrane protein (MP), AdiC. Briefly, AdiC with a 10× Histidine tag on the C-terminal was recombinantly overexpressed using the convenient prokaryotic expression system in BL21(DE3) pLysS cell line and was extracted by solubilizing using n-dodecyl-β-D-maltopyranoside (DDM) and purified by metal affinity chromatography in the same detergent. After confirming the purity by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis), recombinant AdiC was subjected to 8 rounds of conventional Systematic Evolution of Ligands by EXponential enrichment (SELEX) for the in vitro selection of 2′F-Pyrimidine-modified candidate RNA aptamers. Oligonucleotide sequences from the final library of selection were cloned and identified by Sanger sequencing. Three sequences (noorA noorB and noorC) were found abundant among 100 clones and thus chosen as the basis for subsequent informatics analyses. Using a computational approach with comparative analyses of primary and optimal (MFE) 2D secondary structures predicted by RNAfold and Kinefold programs, we were able to identify three conserved motifs (one of which we annotated as a putative local binding domain and the other two as stabilizers) in noorA, B and C and also in two more sequences (AdiC19 and AdiC72) identified in a different phylogenetic family. We also defined a consensus secondary structure to which these five proto-aptamer conform. Subsequent prediction-driven (in-silico) mutational deletions of the stabilizer motifs revealed a global impact on several of the predicted MFE structures in maintaining the substructure that harbors the putative binding motif. The five aptamer candidates generated in this work may have prospects as versatile agents applied in areas of aptamer-based diagnostics and therapeutics where they may be used to assess bacterial infections in patients (e.g. by aptasensors) or as antimicrobial pharmaceutical drugs or drug delivery agents for these infections. At the level of basic scientific research, these aptamers may also be useful in further understanding the structural features and biochemical functions of AdiC. More broadly, given the scarcity of SELEX trials against purified full-length MPs over the past ∼20 years, this thesis presents itself as one of the proof-of-principle studies that bring attention to the usability of purified full-length MPs for the selection of aptamers after MP reconstitution in detergent micelles. This advocacy is in line with the advantages provided by conventional protein-SELEX over cell-SELEX and utility of soluble intra- and extracellular MP domains used as targets. At the end of this thesis, we direct the spotlight on a special opportunity available for complex MPs such as receptors, transporters, and channels that may often be inaccessible for aptamer targeting due to multi-passing or deep integration to and intimacy with their membranes.