A large size-selective DNA nanopore with sensing applications

Abstract

Transmembrane nanostructures like ion channels and transporters perform key biological functions by controlling flow of molecules across lipid bilayers. Much work has gone into engineering artificial nanopores and applications in selective gating of molecules, label-free detection/sensing of biomolecules and DNA sequencing have shown promise. Here, we use DNA origami to create a synthetic 9 nm wide DNA nanopore, controlled by programmable, lipidated flaps and equipped with a size-selective gating system for the translocation of macromolecules. Successful assembly and insertion of the nanopore into lipid bilayers are validated by transmission electron microscopy (TEM), while selective translocation of cargo and the pore mechanosensitivity are studied using optical methods, including single-molecule, total internal reflection fluorescence (TIRF) microscopy. Size-specific cargo translocation and oligonucleotide-triggered opening of the pore are demonstrated showing that the DNA nanopore can function as a real-time detection system for external signals, offering potential for a variety of highly parallelized sensing applications.

Label free fluorescence quantification of hydrolytic enzyme activity on native substrates reveal how lipase function depends on membrane curvature

“…Extraction of fluorescent intensity traces from individual liposomes where done using in-house developed routines in python, following recently published methodologies 38,57 . Briefly, time series were firstly corrected for potential drift from addition of enzyme as recently published 38 . After ensuring that no drift was observed, the full trajectories would be collected and individually corrected for potential background contributions as also shown in 38 , resulting in trajectories as displayed in Fig.…”

Across kingdom biased CYP-mediated metabolism via small-molecule ligands docking on P450 oxidoreductase

“…We therefore hypothesized that POR biased specificity might originate from biased conformational sampling. We used Total Internal Reflection Fluorescence (TIRF) microscopy [29][30][31] to record smFRET traces and directly observe conformational sampling of SbPOR2b and its remodeling by ligands. Data were recorded using the Alternating-Laser Excitation (ALEX) methodology 32 that we and others have been using extensively 28,29,33 .…”

DeepFRET, a software for rapid and automated single-molecule FRET data classification using deep learning

“…The fully automated analysis software operates with minimal crucial human intervention and requires only a threshold on the data quality confidence, as an initial step, to output detailed quantification of structural dynamic from raw images. This is attained by an intuitive and user-friendly interface that integrates and automates common smFRET analysis procedures ( Greenfeld et al, 2012 ; Hellenkamp et al, 2018 ; Hon and Gonzalez, 2019 ; Juette et al, 2016 ; Preus et al, 2015 ; Stella et al, 2018 ) from raw image analysis and background-corrected intensity trace extraction ( Thomsen et al, 2019 ), to sophisticated trace classification, statistical analysis of single-molecule data and production of publication-quality figures of dynamic structural biology insights (see Materials and methods). DeepFRET comes as a free-to-use standalone executable for both Windows and Mac users allowing end-users with limited programming skills to easily operate it.…”