scholarly journals Single-molecule DNA origami aptasensors for real-time biomarker detection

2020 ◽  
Vol 8 (30) ◽  
pp. 6352-6356
Author(s):  
Keitel Cervantes-Salguero ◽  
Mark Freeley ◽  
Jorge L. Chávez ◽  
Matteo Palma
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Conformational Changes ◽  
Dna Origami ◽  
Biomarker Detection ◽  
Dna Nanostructures

DNA nanostructures as platforms to monitor the inherent conformational changes of aptamers upon analyte binding, with single-molecule resolution and real-time capability.

scholarly journals Real-time analysis of RAG complex activity in V(D)J recombination

2016 ◽  
Vol 113 (42) ◽  
pp. 11853-11858 ◽  
Author(s):  
Jennifer Zagelbaum ◽  
Noriko Shimazaki ◽  
Zitadel Anne Esguerra ◽  
Go Watanabe ◽  
Michael R. Lieber ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Conformational Changes ◽  
Signal Sequence ◽  
High Mobility ◽  
Time Analysis ◽  
Complex Activity ◽  
Synaptic Complex ◽  
Single Molecule Fret ◽  
Real Time Analysis

Single-molecule FRET (smFRET) and single-molecule colocalization (smCL) assays have allowed us to observe the recombination-activating gene (RAG) complex reaction mechanism in real time. Our smFRET data have revealed distinct bending modes at recombination signal sequence (RSS)-conserved regions before nicking and synapsis. We show that high mobility group box 1 (HMGB1) acts as a cofactor in stabilizing conformational changes at the 12RSS heptamer and increasing RAG1/2 binding affinity for 23RSS. Using smCL analysis, we have quantitatively measured RAG1/2 dwell time on 12RSS, 23RSS, and non-RSS DNA, confirming a strict RSS molecular specificity that was enhanced in the presence of a partner RSS in solution. Our studies also provide single-molecule determination of rate constants that were previously only possible by indirect methods, allowing us to conclude that RAG binding, bending, and synapsis precede catalysis. Our real-time analysis offers insight into the requirements for RSS–RSS pairing, architecture of the synaptic complex, and dynamics of the paired RSS substrates. We show that the synaptic complex is extremely stable and that heptamer regions of the 12RSS and 23RSS substrates in the synaptic complex are closely associated in a stable conformational state, whereas nonamer regions are perpendicular. Our data provide an enhanced and comprehensive mechanistic description of the structural dynamics and associated enzyme kinetics of variable, diversity, and joining [V(D)J] recombination.

scholarly journals Biophysical characterization of DNA origami nanostructures reveals inaccessibility to intercalation binding sites

2019 ◽  
Author(s):  
Helen L. Miller ◽  
Sonia Contera ◽  
Adam J.M. Wollman ◽  
Adam Hirst ◽  
Katherine E. Dunn ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Single Molecule ◽  
Targeted Drug Delivery ◽  
Binding Sites ◽  
Dna Origami ◽  
Target Cells ◽  
Dna Nanostructures ◽  
Synthetic Dna ◽  
Drug Molecules ◽  
Targeted Drug

AbstractIntercalation of drug molecules into synthetic DNA nanostructures formed through self-assembled origami has been postulated as a valuable future method for targeted drug delivery. This is due to the excellent biocompatibility of synthetic DNA nanostructures, and high potential for flexible programmability including facile drug release into or near to target cells. Such favourable properties may enable high initial loading and efficient release for a predictable number of drug molecules per nanostructure carrier, important for efficient delivery of safe and effective drug doses to minimise non-specific release away from target cells. However, basic questions remain as to how intercalation-mediated loading depends on the DNA carrier structure. Here we use the interaction of dyes YOYO-1 and acridine orange with a tightly-packed 2D DNA origami tile as a simple model system to investigate intercalation-mediated loading. We employed multiple biophysical techniques including single-molecule fluorescence microscopy, atomic force microscopy, gel electrophoresis and controllable damage using low temperature plasma on synthetic DNA origami samples. Our results indicate that not all potential DNA binding sites are accessible for dye intercalation, which has implications for future DNA nanostructures designed for targeted drug delivery.

scholarly journals 48-spot single-molecule FRET setup with periodic acceptor excitation

2017 ◽  
Author(s):  
Antonino Ingargiola ◽  
Maya Segal ◽  
Angelo Gulinatti ◽  
Ivan Rech ◽  
Ivan Labanca ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Conformational Changes ◽  
High Throughput Screening ◽  
Acquisition Time ◽  
Enzymatic Reactions ◽  
Single Molecule Fret ◽  
Array Detectors ◽  
Donor And Acceptor

Single-molecule FRET (smFRET) allows measuring distances between donor and acceptor fluorophores on the 3-10 nm range. Solution-based smFRET allows measurement of binding-unbinding events or conformational changes of dye-labeled biomolecules without ensemble averaging and free from surface perturbations. When employing dual (or multi) laser exci-tation, smFRET allows resolving the number of fluorescent labels on each molecule, greatly enhancing the ability to study heterogeneous samples. A major drawback to solution-based smFRET is the low throughput, which renders repetitive measurements expensive and hin-ders the ability to study kinetic phenomena in real-time.Here we demonstrate a high-throughput smFRET system which multiplexes acquisition by using 48 excitation spots and two 48-pixel SPAD array detectors. The system employs two excitation lasers allowing separation of species with one or two active fluorophores. The performance of the system is demonstrated on a set of doubly-labeled double-stranded DNA oligonucleotides with different distances between donor and acceptor dyes along the DNA duplex. We show that the acquisition time for accurate subpopulation identification is reduced from several minutes to seconds, opening the way to high-throughput screening applications and real-time kinetics studies of enzymatic reactions such as DNA transcription by bacterial RNA polymerase.

scholarly journals Real-time Conformational Dynamics of SARS-CoV-2 Spikes on Virus Particles

2020 ◽  
Author(s):  
Maolin Lu ◽  
Pradeep D. Uchil ◽  
Wenwei Li ◽  
Desheng Zheng ◽  
Daniel S. Terry ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Conformational Changes ◽  
Vaccine Development ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Virus Particles ◽  
Immunogen Design ◽  
Real Time Information

AbstractSARS-CoV-2 spike (S) mediates entry into cells and is critical for vaccine development against COVID-19. Structural studies have revealed distinct conformations of S, but real-time information that connects these structures, is lacking. Here we apply single-molecule Förster Resonance Energy Transfer (smFRET) imaging to observe conformational dynamics of S on virus particles. Virus-associated S dynamically samples at least four distinct conformational states. In response to hACE2, S opens sequentially into the hACE2-bound S conformation through at least one on-path intermediate. Conformational preferences of convalescent plasma and antibodies suggest mechanisms of neutralization involving either competition with hACE2 for binding to RBD or allosteric interference with conformational changes required for entry. Our findings inform on mechanisms of S recognition and conformations for immunogen design.

scholarly journals Single-molecule polarization microscopy of DNA intercalators sheds light on the structure of S-DNA

2019 ◽  
Vol 5 (3) ◽  
pp. eaav1083 ◽  
Author(s):  
Adam S. Backer ◽  
Andreas S. Biebricher ◽  
Graeme A. King ◽  
Gijs J. L. Wuite ◽  
Iddo Heller ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Dna Origami ◽  
Polarization Microscopy ◽  
Base Pairs ◽  
Polarization Imaging ◽  
Dna Intercalators ◽  
Fundamental Information ◽  
Dna Base ◽  
Dna Base Pairs

DNA structural transitions facilitate genomic processes, mediate drug-DNA interactions, and inform the development of emerging DNA-based biotechnology such as programmable materials and DNA origami. While some features of DNA conformational changes are well characterized, fundamental information such as the orientations of the DNA base pairs is unknown. Here, we use concurrent fluorescence polarization imaging and DNA manipulation experiments to probe the structure of S-DNA, an elusive, elongated conformation that can be accessed by mechanical overstretching. To this end, we directly quantify the orientations and rotational dynamics of fluorescent DNA-intercalated dyes. At extensions beyond the DNA overstretching transition, intercalators adopt a tilted (θ ~ 54°) orientation relative to the DNA axis, distinct from the nearly perpendicular orientation (θ ~ 90°) normally assumed at lower extensions. These results provide the first experimental evidence that S-DNA has substantially inclined base pairs relative to those of the standard (Watson-Crick) B-DNA conformation.

Monitoring patterned enzymatic polymerization on DNA origami at single-molecule level

Nanoscale ◽  
2015 ◽  
Vol 7 (25) ◽  
pp. 10970-10973 ◽  
Author(s):  
A. H. Okholm ◽  
H. Aslan ◽  
F. Besenbacher ◽  
M. Dong ◽  
J. Kjems
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Dna Origami ◽  
Enzymatic Polymerization ◽  
Dna Polymerization ◽  
Single Molecule Level ◽  
Site Selective

Site-selective enzymatic polymerization by TdT on surface-bound DNA origami embedded in BSA. DNA polymerization was visualized real time by AFM.

scholarly journals Single molecule analysis of structural fluctuations in DNA nanostructures

Nanoscale ◽  
2019 ◽  
Vol 11 (39) ◽  
pp. 18475-18482 ◽  
Author(s):  
Mette D. E. Jepsen ◽  
Rasmus Schøler Sørensen ◽  
Christopher Maffeo ◽  
Aleksei Aksimentiev ◽  
Jørgen Kjems ◽  
...  
Keyword(s):  
Single Molecule ◽  
Local Structure ◽  
Brownian Dynamics ◽  
Dna Origami ◽  
Coarse Grain ◽  
Dna Nanostructures ◽  
Single Molecule Fret ◽  
Structural Fluctuations ◽  
Single Molecule Analysis

The local structure of DNA origami boxes shows fluctuations, visualized through single molecule FRET and coarse grain Brownian dynamics.

Real-Time Imaging of Single-Molecule Enzyme Cascade Using a DNA Origami Raft

2017 ◽  
Vol 139 (48) ◽  
pp. 17525-17532 ◽  
Author(s):  
Lele Sun ◽  
Yanjing Gao ◽  
Yan Xu ◽  
Jie Chao ◽  
Huajie Liu ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Dna Origami ◽  
Real Time Imaging ◽  
Enzyme Cascade
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