Binding Affinity Measurements Between DNA Aptamers and their Virus Targets Using ELONA and MST.

Aptamers have been selected with strong affinity and high selectivity for a wide range of targets, as recently highlighted by the development of aptamer-based sensors that can differentiate infectious from non-infectious viruses, including human adenovirus and SARS-CoV-2. Accurate determination of the binding affinity between the DNA aptamers and their viral targets is the first step to understanding the molecular recognition of viral particles and the potential uses of aptamers in various diagnostics and therapeutic applications. Here, we describe protocols to obtain the binding curve of the DNA aptamers to SARS-CoV-2 using Enzyme-Linked Oligonucleotide Assay (ELONA) and MicroScale Thermophoresis (MST). These methods allow for the determination of the binding affinity of the aptamer to the infectious SARS-CoV-2 and the selectivity of this aptamer against the same SARS-CoV-2 that has been rendered non-infectious by UV inactivation, and other viruses. Compared to other techniques like Electrophoretic Mobility Shift Assay (EMSA), Surface Plasmon Resonance (SPR), and Isothermal Titration Calorimetry (ITC), these methods have advantages for working with larger particles like viruses and with samples that require biosafety level 2 facilities.

Direct detection of human adenovirus or SARS-CoV-2 with ability to inform infectivity using DNA aptamer-nanopore sensors.

Viral infections are a major global health issue, but no current method allows rapid, direct, and ultrasensitive quantification of intact viruses with the ability to inform infectivity, causing misdiagnoses and spread of the viruses. Here, we report a method for direct detection and differentiation of infectious from noninfectious human adenovirus and SARS-CoV-2, as well as from other virus types, without any sample pretreatment. DNA aptamers are selected from a DNA library to bind intact infectious, but not noninfectious, virus and then incorporated into a solid-state nanopore, which allows strong confinement of the virus to enhance sensitivity down to 1 pfu/ml for human adenovirus and 1 × 104 copies/ml for SARS-CoV-2. Applications of the aptamer-nanopore sensors in different types of water samples, saliva, and serum are demonstrated for both enveloped and nonenveloped viruses, making the sensor generally applicable for detecting these and other emerging viruses of environmental and public health concern.

Recent progress in developing fluorescent probes for imaging cell metabolites.

Cellular metabolites play a crucial role in promoting and regulating cellular activities, but it has been difficult to monitor these cellular metabolites in living cells and in real time. Over the past decades, iterative development and improvements of fluorescent probes have been made, resulting in the effective monitoring of metabolites. In this review, we highlight recent progress in the use of fluorescent probes for tracking some key metabolites, such as adenosine triphosphate, cyclic adenosine monophosphate, cyclic guanosine 5'-monophosphate, Nicotinamide adenine dinucleotide (NADH), reactive oxygen species, sugar, carbon monoxide, and nitric oxide for both whole cell and subcellular imaging.