Ideal‐Filter Capillary Electrophoresis (IFCE) Facilitates the One‐Step Selection of Aptamers

Selection of aptamers from oligonucleotide libraries currently requires multiple rounds of alternating steps of partitioning of binders from nonbinders and enzymatic amplification of all collected oligonucleotides. Herein, we report a highly practical solution for reliable one‐step selection of aptamers. We introduce partitioning by ideal‐filter capillary electrophoresis (IFCE) in which binders and nonbinders move in the opposite directions. The efficiency of IFCE‐based partitioning reaches 10⁹, which is ten million times higher than that of typical solid‐phase partitioning methods. One step of IFCE‐based partitioning is sufficient for the selection of a high‐affinity aptamer pool for a protein target. Partitioning by IFCE promises to become an indispensable tool for fast and robust selection of binders from different types of oligonucleotide libraries.     

Single‐Molecule Monitoring of the Structural Switching Dynamics of Nucleic Acids through Controlling Fluorescence Blinking

Single‐molecule fluorescence resonance energy transfer (smFRET) is a powerful tool to investigate the dynamics of biomolecular events in real time. However, it requires two fluorophores and can be applied only to dynamics that accompany large changes in distance between the molecules. Herein, we introduce a method for kinetic analysis based on control of fluorescence blinking (KACB), a general approach to investigate the dynamics of biomolecules by using a single fluorophore. By controlling the kinetics of the redox reaction the blinking kinetics or pattern can be controlled to be affected by microenvironmental changes around a fluorophore (rKACB), thereby enabling real‐time single‐molecule measurement of the structure‐changing dynamics of nucleic acids.     

In Vitro Selection of Circular DNA Aptamers for Biosensing Applications

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.     

A Biomimetic Phosphatidylcholine‐Terminated Monolayer Greatly Improves the In Vivo Performance of Electrochemical Aptamer‐Based Sensors

The real‐time monitoring of specific analytes in situ in the living body would greatly advance our understanding of physiology and the development of personalized medicine. Because they are continuous (wash‐free and reagentless) and are able to work in complex media (e.g., undiluted serum), electrochemical aptamer‐based (E‐AB) sensors are promising candidates to fill this role. E‐AB sensors suffer, however, from often‐severe baseline drift when deployed in undiluted whole blood either in vitro or in vivo. We demonstrate that cell‐membrane‐mimicking phosphatidylcholine (PC)‐terminated monolayers improve the performance of E‐AB sensors, reducing the baseline drift from around 70 % to just a few percent after several hours in flowing whole blood in vitro. With this improvement comes the ability to deploy E‐AB sensors directly in situ in the veins of live animals, achieving micromolar precision over many hours without the use of physical barriers or active drift‐correction algorithms.