scholarly journals Frontispiece: Single-Molecule Sensing with Nanopore Confinement: From Chemical Reactions to Biological Interactions

2018 ◽  
Vol 24 (50) ◽  
Author(s):  
Yao Lin ◽  
Yi-Lun Ying ◽  
Rui Gao ◽  
Yi-Tao Long
Keyword(s):  
Chemical Reactions ◽  
Single Molecule ◽  
Biological Interactions ◽  
Nanopore Confinement

scholarly journals Automated AFM analysis of DNA bending reveals initial lesion sensing strategies of DNA glycosylases

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Disha M. Bangalore ◽  
Hannah S. Heil ◽  
Christian F. Mehringer ◽  
Lisa Hirsch ◽  
Katherina Hemmen ◽  
...  
Keyword(s):  
Single Molecule ◽  
High Efficiency ◽  
Excision Repair ◽  
Lesion Detection ◽  
Analysis Tool ◽  
Biological Interactions ◽  
Dna Glycosylases ◽  
High Throughput Analysis ◽  
Mutagenic Potential ◽  
Enhanced Sensitivity

Abstract Base excision repair is the dominant DNA repair pathway of chemical modifications such as deamination, oxidation, or alkylation of DNA bases, which endanger genome integrity due to their high mutagenic potential. Detection and excision of these base lesions is achieved by DNA glycosylases. To investigate the remarkably high efficiency in target site search and recognition by these enzymes, we applied single molecule atomic force microscopy (AFM) imaging to a range of glycosylases with structurally different target lesions. Using a novel, automated, unbiased, high-throughput analysis approach, we were able to resolve subtly different conformational states of these glycosylases during DNA lesion search. Our results lend support to a model of enhanced lesion search efficiency through initial lesion detection based on altered mechanical properties at lesions. Furthermore, its enhanced sensitivity and easy applicability also to other systems recommend our novel analysis tool for investigations of diverse, fundamental biological interactions.

scholarly journals Single-molecule nanopore enzymology

2017 ◽  
Vol 372 (1726) ◽  
pp. 20160230 ◽  
Author(s):  
Kherim Willems ◽  
Veerle Van Meervelt ◽  
Carsten Wloka ◽  
Giovanni Maglia
Keyword(s):  
Membrane Proteins ◽  
Chemical Reactions ◽  
Bias Voltage ◽  
Single Molecule ◽  
Ionic Current ◽  
Enzymatic Reactions ◽  
Current Passing ◽  
Single Molecule Level ◽  
Membrane Pores ◽  
Native Proteins

Biological nanopores are a class of membrane proteins that open nanoscale water conduits in biological membranes. When they are reconstituted in artificial membranes and a bias voltage is applied across the membrane, the ionic current passing through individual nanopores can be used to monitor chemical reactions, to recognize individual molecules and, of most interest, to sequence DNA. In addition, a more recent nanopore application is the analysis of single proteins and enzymes. Monitoring enzymatic reactions with nanopores, i.e. nanopore enzymology, has the unique advantage that it allows long-timescale observations of native proteins at the single-molecule level. Here, we describe the approaches and challenges in nanopore enzymology. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

scholarly journals Simple nanofluidic devices for high-throughput, non-equilibrium studies at the single-molecule level

2017 ◽  
Author(s):  
Carel Fijen ◽  
Mattia Fontana ◽  
Serge G. Lemay ◽  
Klaus Mathwig ◽  
Johannes Hohlbein
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Single Particle Tracking ◽  
Biological Interactions ◽  
Dynamic Heterogeneity ◽  
High Throughput Analysis ◽  
Equilibrium Studies ◽  
Single Molecule Level ◽  
Nanofluidic Devices ◽  
And Performance

ABSTRACTSingle-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. Probing chemical and biological interactions and reactions with high throughput and time resolution, however, remains challenging and often requires surface-immobilized entities. Here, utilizing camera-based fluorescence microscopy, we present glass-made nanofluidic devices in which fluorescently labelled molecules flow through nanochannels that confine their diffusional movement. The first design features an array of parallel nanochannels for high-throughput analysis of molecular species under equilibrium conditions allowing us to record 200.000 individual localization events in just 10 minutes. Using these localizations for single particle tracking, we were able to obtain accurate flow profiles including flow speeds and diffusion coefficients inside the channels.A second design featuring a T-shaped nanochannel enables precise mixing of two different species as well as the continuous observation of chemical reactions. We utilized the design to visualize enzymatically driven DNA synthesis in real time and at the single-molecule level. Based on our results, we are convinced that the versatility and performance of the nanofluidic devices will enable numerous applications in the life sciences.

Modern Biophysical Approaches to Study Protein–Ligand Interactions

2018 ◽  
Vol 13 (04) ◽  
pp. 133-155
Author(s):  
Priyanka Biswas
Keyword(s):  
Single Molecule ◽  
Analytical Ultracentrifugation ◽  
Correlation Spectroscopy ◽  
Titration Calorimetry ◽  
Resonance Fluorescence ◽  
Biological Interactions ◽  
Protein Ligand Interactions ◽  
Dual Polarization Interferometry ◽  
Ligand Interactions

Protein–ligand interactions act as a pivot to the understanding of most of the biological interactions. The study of interactions between proteins and cellular molecules has led to the establishment and identification of various important pathways that control biological systems. Investigators working in different fields of biological sciences have an intrinsic interest in this field and complement their findings by the application of different biophysical approaches and tools to quantify protein–ligand interactions that include protein–small molecules, protein–DNA, protein–RNA, protein–protein both in vitro and in vivo. In this paper, the various biophysical techniques that can be employed to study such interactions will be discussed. In addition to native gel electrophoresis and fluorescence-based methods, more details will be discussed, on the broad range of modern day biophysical tools such as Circular Dichroism, Fourier Transform Infrared (FTIR) Spectroscopy, Isothermal Titration Calorimetry, Analytical Ultracentrifugation, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy, Differential Scanning Fluorimetry, Nuclear Magnetic Resonance, Mass Spectroscopy, Single Molecule Spectroscopy, Dual Polarization Interferometry, Micro Scale Thermophoresis and Electro–switchable Biosensors that can be used to study the different aspects of protein–ligand interactions.

Single-molecule chemical reactions on DNA origami

2010 ◽  
Vol 5 (3) ◽  
pp. 200-203 ◽  
Author(s):  
Niels V. Voigt ◽  
Thomas Tørring ◽  
Alexandru Rotaru ◽  
Mikkel F. Jacobsen ◽  
Jens B. Ravnsbæk ◽  
...  
Keyword(s):  
Chemical Reactions ◽  
Single Molecule ◽  
Dna Origami
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