In contrast to sequence-specific techniques such as polymerase chain reaction, DNA sequencing does not require prior knowledge of the sample for surveying DNA. However, current sequencing technologies demand high inputs for a suitable library preparation, which typically necessitates DNA amplification, even for single-molecule sequencing methods. Here, electro-optical zero-mode waveguides (eZMWs) are presented, which can load DNA into the confinement of zero-mode waveguides with high efficiency and negligible DNA fragment length bias. Using eZMWs, highly efficient voltage-induced loading of DNA fragments of various sizes from ultralow inputs (nanogram-to-picogram levels) is observed. Rapid DNA fragment identification is demonstrated by burst sequencing of short and long DNA molecules (260 and 20 000 bp) loaded from an equimolar picomolar-level concentration mixture in just a few minutes. The device allows further studies in which low-input DNA capture is essential, for example, in epigenetics, where native DNA is required for obtaining modified base information.
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http://dx.doi.org/10.1002/adma.202108479 | DOI Listing |
QRB Discov
December 2024
Department of Chemistry, University of Basel, Basel, Switzerland.
Single Molecule Förster Resonance Energy Transfer (smFRET) is a popular technique to directly observe biomolecular dynamics in real time, offering unique mechanistic insight into proteins, ribozymes, and so forth. However, inevitable photobleaching of the fluorophores puts a stringent limit on the total time a surface-tethered molecule can be monitored, fundamentally limiting the information gain through conventional smFRET measurements. DyeCycling addresses this problem by using reversibly - instead of covalently - coupled FRET fluorophores, through which it can break the photobleaching limit and theoretically provide unlimited observation time.
View Article and Find Full Text PDFSci Rep
September 2024
Department of Optics and Photonics, National Central University, No. 300, Zhongda Rd., Zhongli, Taoyuan, 320317, Taiwan.
We refresh the design of zero-mode waveguides (ZMWs) by introducing metamaterials that makes the zeroth order resonant mode existence. Of particular importance, the resulting electromagnetic field exhibits nearly constant distribution but not a trivial solution of Maxwell's equation, showing great advantage to equalize the excitation rate of molecules throughout the waveguides. A closed form expression for the wave impedance is derived which is verified by the finite-difference time-domain simulations.
View Article and Find Full Text PDFACS Appl Mater Interfaces
May 2024
School of Biomedical Engineering (Suzhou), Division of Life Sciences and Medicine, University of Science and Technology of China, 230026 Hefei, China.
Single-molecule detection with high accuracy and specialty plays an important role in biomedical diagnosis and screening. Zero-mode waveguides (ZMWs) enable the possibility of single biological molecule detection in real time. Nevertheless, the absence of a reliable assessment for single effective complex loading has constrained further applications of ZMWs in complex interaction.
View Article and Find Full Text PDFMethods Mol Biol
April 2024
Plant Functional Genomics Lab, Institute of Molecular Biotechnology, Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria.
PacBio long-read sequencing is a third-generation technology that generates long reads up to 20 kilobases (kb), unlike short-read sequencing instruments that produce up to 600 bases. Long-read sequencing is particularly advantageous in higher organisms, such as humans and plants, where repetitive regions in the genome are more abundant. The PacBio long-read sequencing uses a single molecule, real-time approach where the SMRT cells contain several zero-mode waveguides (ZMWs).
View Article and Find Full Text PDFProc Natl Acad Sci U S A
February 2024
Institute of Physiology II, Jena University Hospital, Friedrich Schiller University, Jena 07743, Germany.
The cooperative action of the subunits in oligomeric receptors enables fine-tuning of receptor activation, as demonstrated for the regulation of voltage-activated HCN pacemaker ion channels by relating cAMP binding to channel activation in ensemble signals. HCN channels generate electric rhythmicity in specialized brain neurons and cardiomyocytes. There is conflicting evidence on whether binding cooperativity does exist independent of channel activation or not, as recently reported for detergent-solubilized receptors positioned in zero-mode waveguides.
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