Nanoscale “Curtain Rods”: High-Throughput Tools for Studying DNA-Protein Interactions

2008 ◽  
Vol 1138 ◽  
Author(s):  
Teresa Fazio ◽  
Mari-Liis Visnapuu ◽  
Shalom J. Wind ◽  
Eric Greene
Keyword(s):  
Data Acquisition ◽  
Real Time ◽  
Lipid Bilayer ◽  
High Throughput ◽  
Single Molecule ◽  
Protein Interactions ◽  
Massively Parallel ◽  
Dna Interactions ◽  
Parallel Data ◽  
Protein Dna Interactions

AbstractIn this work, we combine nanoscale engineering with single-molecule biology to probe the biochemical interactions between individual proteins and DNA. This approach, a vast improvement over previous methods, constructs a platform to observe thousands of protein-DNA interactions in real time with unprecedented detail. A key challenge in these experiments involves collecting enough statistically relevant data in order to analyze reactions which are designed to be probed individually. “DNA curtains” are formed by flowing the DNA tethered to a lipid bilayer across nanopatterned barriers, facilitating massively parallel data acquisition.

scholarly journals Massively parallel single-molecule telomere length measurement with digital real-time PCR

2020 ◽  
Vol 6 (34) ◽  
pp. eabb7944 ◽  
Author(s):  
Yongqiang Luo ◽  
Ramya Viswanathan ◽  
Manoor Prakash Hande ◽  
Amos Hong Pheng Loh ◽  
Lih Feng Cheow
Keyword(s):  
Telomere Length ◽  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Real Time Pcr ◽  
Length Distribution ◽  
Telomere Maintenance ◽  
Massively Parallel ◽  
Primary Tumors ◽  
Absolute Length

Telomere length is a promising biomarker for age-associated diseases and cancer, but there are still substantial challenges to routine telomere analysis in clinics because of the lack of a simple and rapid yet scalable method for measurement. We developed the single telomere absolute-length rapid (STAR) assay, a novel high-throughput digital real-time PCR approach for rapidly measuring the absolute lengths and quantities of individual telomere molecules. We show that this technique provides the accuracy and sensitivity to uncover associations between telomere length distribution and telomere maintenance mechanisms in cancer cell lines and primary tumors. The results indicate that the STAR assay is a powerful tool to enable the use of telomere length distribution as a biomarker in disease and population-wide studies.

scholarly journals DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level

Applied Nano ◽  
2022 ◽  
Vol 3 (1) ◽  
pp. 16-41
Author(s):  
Aurimas Kopūstas ◽  
Mindaugas Zaremba ◽  
Marijonas Tutkus
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Dna Interaction ◽  
Dna Interactions ◽  
Ensemble Averaging ◽  
Target Search ◽  
Single Molecule Level ◽  
Protein Dna Interactions ◽  
Long Time

Protein-DNA interactions are the core of the cell’s molecular machinery. For a long time, conventional biochemical methods served as a powerful investigatory basis of protein-DNA interactions and target search mechanisms. Currently single-molecule (SM) techniques have emerged as a complementary tool for studying these interactions and have revealed plenty of previously obscured mechanistic details. In comparison to the traditional ones, SM methods allow direct monitoring of individual biomolecules. Therefore, SM methods reveal reactions that are otherwise hidden by the ensemble averaging observed in conventional bulk-type methods. SM biophysical techniques employing various nanobiotechnology methods for immobilization of studied molecules grant the possibility to monitor individual reaction trajectories of biomolecules. Next-generation in vitro SM biophysics approaches enabling high-throughput studies are characterized by much greater complexity than the ones developed previously. Currently, several high-throughput DNA flow-stretch assays have been published and have shown many benefits for mechanistic target search studies of various DNA-binding proteins, such as CRISPR-Cas, Argonaute, various ATP-fueled helicases and translocases, and others. This review focuses on SM techniques employing surface-immobilized and relatively long DNA molecules for studying protein-DNA interaction mechanisms.

scholarly journals High-throughput single-molecule studies of protein-DNA interactions

FEBS Letters ◽  
2014 ◽  
Vol 588 (19) ◽  
pp. 3539-3546 ◽  
Author(s):  
Aaron D. Robison ◽  
Ilya J. Finkelstein
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Dna Interactions ◽  
Protein Dna Interactions ◽  
Single Molecule Studies

Real-time, single-molecule observation of biomolecular interactions inside nanophotonic zero mode waveguides

2021 ◽  
Author(s):  
Anastasiia Nemashkalo ◽  
Mary Elizabeth Phipps ◽  
Scott Patrick Hennelly ◽  
Peter Marvin Goodwin
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Ribosomal Rna ◽  
Protein Interactions ◽  
Zero Mode ◽  
Single Molecule Level ◽  
Sequencing By Synthesis ◽  
Time Observation ◽  
Real Time Observation

Abstract Living cells rely on numerous protein-protein, RNA-protein and DNA-protein interactions for processes such as gene expression, biomolecular assembly, protein and RNA degradation. Single-molecule microscopy and spectroscopy are ideal tools for real-time observation and quantification of nucleic acids-protein and protein-protein interactions. One of the major drawbacks of conventional single-molecule imaging methods is low throughput. Methods such as sequencing by synthesis utilizing nanofabrication and single-molecule spectroscopy have brought high throughput into the realm of single-molecule biology. The Pacific Biosceinces RS2 sequencer utilizes sequencing by synthesis within nanophotonic zero mode waveguides. A number of years ago this instrument was unlocked by Pacific Biosciences for custom use by researchers allowing them to monitor biological interactions at the single-molecule level with high throughput. In this capability letter we demonstrate the use of the RS2 sequencer for real time observation of DNA-to-RNA transcription and RNA-protein interactions. We use a relatively complex model – transcription of structured ribosomal RNA from E. coli and interactions of ribosomal RNA with ribosomal proteins. We also show evidence of observation of transcriptional pausing without the application of an external force (as is required for single-molecule pausing studies using optical traps). Overall, in the unlocked, custom mode, the RS2 sequencer can be used to address a wide variety of biological assembly and interaction questions at the single-molecule level with high throughput. This instrument is available for use at the Center for Integrated Nanotechnologies Gateway located at Los Alamos National Laboratory.

DiMeLo-seq: Directed Methylation with Long-read sequencing v2

2021 ◽  
Author(s):  
Nicolas Altemose ◽  
Annie Maslan ◽  
Owen Smith ◽  
Kousik Sundararajan ◽  
Rachel Brown ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Pcr Amplification ◽  
Dna Interaction ◽  
Dna Interactions ◽  
Single Molecule Level ◽  
Multiple Protein ◽  
Protein Dna Interactions ◽  
Adenine Methylation ◽  
Long Read

Directed Methylation and Long-read sequencing (DiMeLo-seq) is a powerful method to map protein-DNA interactions at a single-molecule level across the genome (including repetitive regions). It can be multiplexed to analyze multiple base modifications at once (e.g. endogenous CpG methylation and directed pA-Hia5 adenine methylation). Additionally, PCR amplification is not necessary for this protocol, which means that sequencing readout is proportional to protein-DNA interaction frequency. Finally, DiMeLo-seq can be used to map multiple protein interactions across a long single molecule.

Highly Sensitive Detection of Paraquat with Pillar[5]arene as an aptamer in α-Hemolysin Nanopore

2021 ◽  
Author(s):  
Xiaojia Jiang ◽  
Mingsong Zang ◽  
Fei Li ◽  
Chunxi Hou ◽  
Quan Luo ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Single Molecule Detection ◽  
Sensitive Detection ◽  
High Throughput Analysis ◽  
Throughput Analysis ◽  
The Real ◽  
Highly Sensitive ◽  
Highly Sensitive Detection

Biological nanopore-based techniques have attracted more and more attention recently in the field of single-molecule detection, because they allow the real-time, sensitive, high-throughput analysis. Herein, we report an engineered biological...

scholarly journals EndoBind detects endogenous protein-protein interactions in real time

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Anke Bill ◽  
Sheryll Espinola ◽  
Daniel Guthy ◽  
Jacob R. Haling ◽  
Mylene Lanter ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Protein Interactions ◽  
Continuous Monitoring ◽  
Protein Protein Interactions ◽  
Dimer Formation ◽  
Endogenous Protein ◽  
Extended Duration ◽  
Substrate Addition

AbstractWe present two high-throughput compatible methods to detect the interaction of ectopically expressed (RT-Bind) or endogenously tagged (EndoBind) proteins of interest. Both approaches provide temporal evaluation of dimer formation over an extended duration. Using examples of the Nrf2-KEAP1 and the CRAF-KRAS-G12V interaction, we demonstrate that our method allows for the detection of signal for more than 2 days after substrate addition, allowing for continuous monitoring of endogenous protein-protein interactions in real time.

Sign in / Sign up

Export Citation Format

Share Document