Recent Progress in the Identification of Aptamers Against Bacterial Origins and Their Diagnostic Applications

Aptamers have gained an increasing role as the molecular recognition element (MRE) in diagnostic assay development, since their first conception thirty years ago. The process to screen for nucleic acid-based binding elements (aptamers) was first described in 1990 by the Gold Laboratory. In the last three decades, many aptamers have been identified for a wide array of targets. In particular, the number of reports on investigating single-stranded DNA (ssDNA) aptamer applications in biosensing and diagnostic platforms have increased significantly in recent years. This review article summarizes the recent (2015 to 2020) progress of ssDNA aptamer research on bacteria, proteins, and lipids of bacterial origins that have implications for human infections. The basic process of aptamer selection, the principles of aptamer-based biosensors, and future perspectives will also be discussed.

Modulation of Measles Virus NTAIL Interactions through Fuzziness and Sequence Features of Disordered Binding Sites

In this paper we review our recent findings on the different interaction mechanisms of the C-terminal domain of the nucleoprotein (N) of measles virus (MeV) NTAIL, a model viral intrinsically disordered protein (IDP), with two of its known binding partners, i.e., the C-terminal X domain of the phosphoprotein of MeV XD (a globular viral protein) and the heat-shock protein 70 hsp70 (a globular cellular protein). The NTAIL binds both XD and hsp70 via a molecular recognition element (MoRE) that is flanked by two fuzzy regions. The long (85 residues) N-terminal fuzzy region is a natural dampener of the interaction with both XD and hsp70. In the case of binding to XD, the N-terminal fuzzy appendage of NTAIL reduces the rate of α-helical folding of the MoRE. The dampening effect of the fuzzy appendage on XD and hsp70 binding depends on the length and fuzziness of the N-terminal region. Despite this similarity, NTAIL binding to XD and hsp70 appears to rely on completely different requirements. Almost any mutation within the MoRE decreases XD binding, whereas many of them increase the binding to hsp70. In addition, XD binding is very sensitive to the α-helical state of the MoRE, whereas hsp70 is not. Thus, contrary to hsp70, XD binding appears to be strictly dependent on the wild-type primary and secondary structure of the MoRE.

Selection of Single-Stranded DNA Molecular Recognition Elements against Exotoxin A Using a Novel Decoy-SELEX Method and Sensitive Detection of Exotoxin A in Human Serum

Exotoxin A is one of the virulence factors ofPseudomonas aeruginosa, a bacterium that can cause infections resulting in adverse health outcomes and increased burden to health care systems. Current methods of diagnosingP. aeruginosainfections are time consuming and can require significant preparation of patient samples. This study utilized a novel variation of the Systematic Evolution of Ligand by Exponential Enrichment, Decoy-SELEX, to identify an Exotoxin A specific single-stranded DNA (ssDNA) molecular recognition element (MRE). Its emphasis is on increasing stringency in directing binding toward free target of interest and at the same time decreasing binding toward negative targets. A ssDNA MRE with specificity and affinity was identified after fourteen rounds of Decoy-SELEX. Utilizing surface plasmon resonance measurements, the determined equilibrium dissociation constant(Kd)of the MRE is between 4.2 µM and 4.5 µM, and is highly selective for Exotoxin A over negative targets. A ssDNA MRE modified sandwich enzyme-linked immunosorbent assay (ELISA) has been developed and achieved sensitive detection of Exotoxin A at nanomolar concentrations in human serum. This study has demonstrated the proof-of-principle of using a ssDNA MRE as a clinical diagnostic tool.

In VitroSelection of a Single-Stranded DNA Molecular Recognition Element againstClostridium difficileToxin B and Sensitive Detection in Human Fecal Matter

Toxin B is one of the major virulence factors ofClostridium difficile, a bacterium that is responsible for a significant number of diarrhea cases in acute care settings. Due to the prevalence ofC. difficileinduced diarrhea, rapid and correct diagnosis is crucial in the disease management. In this study, we have employed a stringentin vitroselection method to identify single-stranded DNA molecular recognition elements (MRE) specific for toxin B. At the end of the 12-round selection, one MRE with high affinity(Kd=47.3 nM) for toxin B was identified. The selected MRE demonstrated low cross binding activities on negative targets: bovine serum albumin,Staphylococcus aureusalpha toxin,Pseudomonas aeruginosaexotoxin A, and cholera toxin ofVibrio cholera. A modified sandwich ELISA assay was developed utilizing the selected ssDNA MRE as the antigen capturing element and achieved a sensitive detection of 50 nM of toxin B in human fecal preparations.

Electrogenerated chemiluminescence peptide-based bioassay

Electrogenerated chemiluminescence (ECL) involves the generation of species at electrode surfaces that then undergo electron-transfer reactions to form excited states that emit light. The ECL method is becoming an increasingly promising method in the life sciences, environmental analysis, and clinical analysis, owing to its good selectivity, high sensitivity, wide dynamic concentration response range, and potential and spatial controllability. Here, we give a mini-review on the advances of the ECL peptide-based bioassay using peptide as the molecular recognition element. First, a general history of ECL development is presented. Then, the general strategies of the ECL peptide-based bioassay are reviewed. Particular attention is paid to the related progress in the last 5 years. Finally, we conclude with the future challenges and prospects in the development of the ECL peptide-based bioassay.