scholarly journals Protein-Assisted Room-Temperature Assembly of Rigid, Immobile Holliday Junctions and Hierarchical DNA Nanostructures

Molecules ◽  
2020 ◽  
Vol 25 (21) ◽  
pp. 5099
Author(s):  
Saminathan Ramakrishnan ◽  
Sivaraman Subramaniam ◽  
Charlotte Kielar ◽  
Guido Grundmeier ◽  
A. Francis Stewart ◽  
...  
Keyword(s):  
Room Temperature ◽  
Genetic Recombination ◽  
Protein Complexes ◽  
Dna Nanotechnology ◽  
Holliday Junctions ◽  
Dna Nanostructures ◽  
Fundamental Building Block ◽  
In Vitro Investigation ◽  
Functional Dna

Immobile Holliday junctions represent not only the most fundamental building block of structural DNA nanotechnology but are also of tremendous importance for the in vitro investigation of genetic recombination and epigenetics. Here, we present a detailed study on the room-temperature assembly of immobile Holliday junctions with the help of the single-strand annealing protein Redβ. Individual DNA single strands are initially coated with protein monomers and subsequently hybridized to form a rigid blunt-ended four-arm junction. We investigate the efficiency of this approach for different DNA/protein ratios, as well as for different DNA sequence lengths. Furthermore, we also evaluate the potential of Redβ to anneal sticky-end modified Holliday junctions into hierarchical assemblies. We demonstrate the Redβ-mediated annealing of Holliday junction dimers, multimers, and extended networks several microns in size. While these hybrid DNA–protein nanostructures may find applications in the crystallization of DNA–protein complexes, our work shows the great potential of Redβ to aid in the synthesis of functional DNA nanostructures under mild reaction conditions.

scholarly journals Genetically Encoding Ultra-Stable Virus-like Particles Encapsulating Functional DNA Nanostructures in Living Bacteria

2020 ◽  
Author(s):  
Shai Zilberzwige-Tal ◽  
Dan Alon ◽  
Danielle Gazit ◽  
Shahar Zachariah ◽  
Amit Hollander ◽  
...  
Keyword(s):  
Dna Nanotechnology ◽  
Nucleic Acid Content ◽  
Dna Nanostructures ◽  
Loss Of Function ◽  
Virus Like Particles ◽  
Bio Imaging ◽  
Functional Dna ◽  
Biological Environment

Abstract DNA nanotechnology is leading the field of in vitro molecular-scale device engineering, accumulating to a dazzling array of applications from zeolite-like catalysts to bio-imaging. However, while DNA nanostructures' function is robust under in vitro settings, their implementation in real-world conditions requires overcoming their rapid degradation and subsequent loss of function. Viruses are incredibly sophisticated supramolecular assemblies, able to protect their nucleic acid content in the relatively inhospitable biological environment. Inspired by this natural ability, we engineered both in vitro and in vivo technologies, enabling the encapsulation and protection of functional DNA nanostructures inside MS2 bacteriophage virus-like particles (VLPs). We demonstrate the ssDNA-VLPs nanocomposites (NCs) abilities to encapsulate single-stranded-DNA (ssDNA) of an unprecedented variety of sizes (200–1500 nucleotides (nt)), sequences, and structures while retaining their functionality. Moreover, by exposing these NCs to hostile biological conditions, such as human blood serum, we exhibit that the VLPs serves as an excellent protective shell. To the best of our knowledge, these engineered NCs pose key properties that are yet unattainable by current fabrication methods.

Formation, translocation and resolution of Holliday junctions during homologous genetic recombination

1995 ◽  
Vol 347 (1319) ◽  
pp. 21-25 ◽  
Keyword(s):  
Molecular Mechanisms ◽  
Genetic Recombination ◽  
Holliday Junctions ◽  
The Novel ◽  
E Coli ◽  
The Past ◽  
Endonucleolytic Cleavage ◽  
Insight Into ◽  
Made In

Over the past three or four years, great strides have been made in our understanding of the proteins involved in recombination and the mechanisms by which recombinant molecules are formed. This review summarizes our current understanding of the process by focusing on recent studies of proteins involved in the later steps of recombination in bacteria. In particular, biochemical investigation of the in vitro properties of the E. coli RuvA, RuvB and RuvC proteins have provided our first insight into the novel molecular mechanisms by which Holliday junctions are moved along DNA and then resolved by endonucleolytic cleavage.

scholarly journals The Art of Designing DNA Nanostructures with CAD Software

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2287
Author(s):  
Martin Glaser ◽  
Sourav Deb ◽  
Florian Seier ◽  
Amay Agrawal ◽  
Tim Liedl ◽  
...  
Keyword(s):  
Human Error ◽  
Dna Nanotechnology ◽  
Holliday Junctions ◽  
Dna Origami ◽  
Dna Nanostructures ◽  
Dna Structures ◽  
Current State ◽  
Software Packages ◽  
Wide Range ◽  
Time Required

Since the arrival of DNA nanotechnology nearly 40 years ago, the field has progressed from its beginnings of envisioning rather simple DNA structures having a branched, multi-strand architecture into creating beautifully complex structures comprising hundreds or even thousands of unique strands, with the possibility to exactly control the positions down to the molecular level. While the earliest construction methodologies, such as simple Holliday junctions or tiles, could reasonably be designed on pen and paper in a short amount of time, the advent of complex techniques, such as DNA origami or DNA bricks, require software to reduce the time required and propensity for human error within the design process. Where available, readily accessible design software catalyzes our ability to bring techniques to researchers in diverse fields and it has helped to speed the penetration of methods, such as DNA origami, into a wide range of applications from biomedicine to photonics. Here, we review the historical and current state of CAD software to enable a variety of methods that are fundamental to using structural DNA technology. Beginning with the first tools for predicting sequence-based secondary structure of nucleotides, we trace the development and significance of different software packages to the current state-of-the-art, with a particular focus on programs that are open source.

scholarly journals Bridging-induced phase separation induced by cohesin SMC protein complexes

2021 ◽  
Vol 7 (7) ◽  
pp. eabe5905
Author(s):  
Je-Kyung Ryu ◽  
Céline Bouchoux ◽  
Hon Wing Liu ◽  
Eugene Kim ◽  
Masashi Minamino ◽  
...  
Keyword(s):  
Phase Separation ◽  
Genome Organization ◽  
Fluorescence Recovery After Photobleaching ◽  
Protein Complexes ◽  
Yeast Cells ◽  
Base Pairs ◽  
Fundamental Building Block ◽  
Structural Maintenance Of Chromosome

Structural maintenance of chromosome (SMC) protein complexes are able to extrude DNA loops. While loop extrusion constitutes a fundamental building block of chromosomes, other factors may be equally important. Here, we show that yeast cohesin exhibits pronounced clustering on DNA, with all the hallmarks of biomolecular condensation. DNA-cohesin clusters exhibit liquid-like behavior, showing fusion of clusters, rapid fluorescence recovery after photobleaching and exchange of cohesin with the environment. Strikingly, the in vitro clustering is DNA length dependent, as cohesin forms clusters only on DNA exceeding 3 kilo–base pairs. We discuss how bridging-induced phase separation, a previously unobserved type of biological condensation, can explain the DNA-cohesin clustering through DNA-cohesin-DNA bridges. We confirm that, in yeast cells in vivo, a fraction of cohesin associates with chromatin in a manner consistent with bridging-induced phase separation. Biomolecular condensation by SMC proteins constitutes a new basic principle by which SMC complexes direct genome organization.

scholarly journals In vivo cloning of artificial DNA nanostructures

2008 ◽  
Vol 105 (46) ◽  
pp. 17626-17631 ◽  
Author(s):  
Chenxiang Lin ◽  
Sherri Rinker ◽  
Xing Wang ◽  
Yan Liu ◽  
Nadrian C. Seeman ◽  
...  
Keyword(s):  
Dna Nanotechnology ◽  
Dna Nanostructures ◽  
Scientific Enterprise ◽  
Dna Nanostructure ◽  
Copy Numbers ◽  
Artificial Dna ◽  
Difficult Challenge ◽  
Endonuclease Vii

Mimicking nature is both a key goal and a difficult challenge for the scientific enterprise. DNA, well known as the genetic-information carrier in nature, can be replicated efficiently in living cells. Today, despite the dramatic evolution of DNA nanotechnology, a versatile method that replicates artificial DNA nanostructures with complex secondary structures remains an appealing target. Previous success in replicating DNA nanostructures enzymatically in vitro suggests that a possible solution could be cloning these nanostructures by using viruses. Here, we report a system where a single-stranded DNA nanostructure (Holliday junction or paranemic cross-over DNA) is inserted into a phagemid, transformed into XL1-Blue cells and amplified in vivo in the presence of helper phages. High copy numbers of cloned nanostructures can be obtained readily by using standard molecular biology techniques. Correct replication is verified by a number of assays including nondenaturing PAGE, Ferguson analysis, endonuclease VII digestion, and hydroxyl radical autofootprinting. The simplicity, efficiency, and fidelity of nature are fully reflected in this system. UV-induced psoralen cross-linking is used to probe the secondary structure of the inserted junction in infected cells. Our data suggest the possible formation of the immobile four-arm junction in vivo.

scholarly journals A histone octamer blocks branch migration of a Holliday junction.

1997 ◽  
Vol 17 (12) ◽  
pp. 7139-7150 ◽  
Author(s):  
M Grigoriev ◽  
P Hsieh
Keyword(s):  
Genetic Recombination ◽  
Intrinsic Rate ◽  
Holliday Junctions ◽  
Micrococcal Nuclease ◽  
Holliday Junction ◽  
Sequence Motif ◽  
Branch Migration ◽  
Histone Octamers ◽  
Nuclease Mapping

The Holliday junction is a key intermediate in genetic recombination. Here, we examine the effect of a nucleosome core on movement of the Holliday junction in vitro by spontaneous branch migration. Histone octamers consisting of H2A, H2B, H3, and H4 are reconstituted onto DNA duplexes containing an artificial nucleosome-positioning sequence consisting of a tandem array of an alternating AT-GC sequence motif. Characterization of the reconstituted branch migration substrates by micrococcal nuclease mapping and exonuclease III and hydroxyl radical footprinting reveal that 70% of the reconstituted octamers are positioned near the center of the substrate and the remaining 30% are located at the distal end, although in both cases some translational degeneracy is observed. Branch migration assays with the octamer-containing substrates reveal that the Holliday junction cannot migrate spontaneously through DNA organized into a nucleosomal core unless DNA-histone interactions are completely disrupted. Similar results are obtained with branch migration substrates containing an octamer positioned on a naturally occurring sequence derived from the yeast GLN3 locus. Digestion of Holliday junctions with T7 endonuclease I establishes that the junction is not trapped by the octamer but can branch migrate in regions free of histone octamers. Our findings suggest that migration of Holliday junctions during recombination and the recombinational repair of DNA damage requires proteins not only to accelerate the intrinsic rate of branch migration but also to facilitate the passage of the Holliday junction through a nucleosome.

Structural DNA nanotechnology: Immobile Holliday junctions to artificial robots

2022 ◽  
Vol 22 ◽  
Author(s):  
Raghu Pradeep Narayanan ◽  
Leeza Abraham
Keyword(s):  
Dna Nanotechnology ◽  
Building Blocks ◽  
Holliday Junctions ◽  
Dna Origami ◽  
Building Structures ◽  
Dna Nanostructures ◽  
3 Dimensional ◽  
Scientific World ◽  
2D And 3D ◽  
Structural Dna Nanotechnology

Abstreact: DNA nanotechnology marvels the scientific world with its capabilities to design, engineer, and demonstrate nanoscale shapes. This review is a condensed version walking the reader through the structural developments in the field over the past 40 years starting from the basic design rules of the double-stranded building block to the most recent advancements in self-assembled hierarchically achieved structures to date. It builds off from the fundamental motivation of building 3-dimensional (3D) lattice structures of tunable cavities going all the way up to artificial nanorobots fighting cancer. The review starts by covering the most important developments from the fundamental bottom-up approach of building structures, which is the ‘tile’ based approach covering 1D, 2D, and 3D building blocks, after which, the top-down approach using DNA origami and DNA bricks is also covered. Thereafter, DNA nanostructures assembled using not so commonly used (yet promising) techniques like i-motifs, quadruplexes, and kissing loops are covered. Highlights from the field of dynamic DNA nanostructures have been covered as well, walking the reader through the various approaches used within the field to achieve movement. The article finally concludes by giving the authors a view of what the future of the field might look like while suggesting in parallel new directions that fellow/future DNA nanotechnologists could think about.

Electron Microscopy and image analysis of nucleo-protein complexes

1994 ◽  
Vol 52 ◽  
pp. 88-89
Author(s):  
E. H. Egelman ◽  
X. Yu
Keyword(s):  
Electron Microscopy ◽  
Image Analysis ◽  
Normal Cell ◽  
Genetic Recombination ◽  
Protein Complexes ◽  
Chromosomal Rearrangements ◽  
Reca Protein ◽  
E Coli ◽  
Helical Polymer ◽  
Specific Protease

The RecA protein of E. coli has been shown to mediate genetic recombination, regulate its own synthesis, control the expression of other genes, act as a specific protease, form a helical polymer and have an ATPase activity, among other observed properties. The unusual filament formed by the RecA protein on DNA has not previously been shown to exist outside of bacteria. Within this filament, the 36 Å pitch of B-form DNA is extended to about 95 Å, the pitch of the RecA helix. We have now establishedthat similar nucleo-protein complexes are formed by bacteriophage and yeast proteins, and availableevidence suggests that this structure is universal across all of biology, including humans. Thus, understanding the function of the RecA protein will reveal basic mechanisms, in existence inall organisms, that are at the foundation of general genetic recombination and repair.Recombination at this moment is assuming an importance far greater than just pure biology. The association between chromosomal rearrangements and neoplasms has become stronger and stronger, and these rearrangements are most likely products of the recombinatory apparatus of the normal cell. Further, damage to DNA appears to be a major cause of cancer.

In Situ Localization of PPAR Gamma and Uncoupling Protein in Mouse Embryo Sections Using Digoxigenin-Labeled Riboprobes

1996 ◽  
Vol 54 ◽  
pp. 32-33
Author(s):  
C. Jennermann ◽  
S. A. Kliewer ◽  
D. C. Morris
Keyword(s):  
Alkaline Phosphatase ◽  
Room Temperature ◽  
Uncoupling Protein ◽  
Ppar Gamma ◽  
Nuclear Hormone Receptor ◽  
Full Length ◽  
Northern Analysis ◽  
Peroxisome Proliferator

Peroxisome proliferator-activated receptor gamma (PPARg) is a member of the nuclear hormone receptor superfamily and has been shown in vitro to regulate genes involved in lipid metabolism and adipocyte differentiation. By Northern analysis, we and other researchers have shown that expression of this receptor predominates in adipose tissue in adult mice, and appears first in whole-embryo mRNA at 13.5 days postconception. In situ hybridization was used to find out in which developing tissues PPARg is specifically expressed.Digoxigenin-labeled riboprobes were generated using the Genius™ 4 RNA Labeling Kit from Boehringer Mannheim. Full length PPAR gamma, obtained by PCR from mouse liver cDNA, was inserted into pBluescript SK and used as template for the transcription reaction. Probes of average size 200 base pairs were made by partial alkaline hydrolysis of the full length transcripts. The in situ hybridization assays were performed as described previously with some modifications. Frozen sections (10 μm thick) of day 18 mouse embryos were cut, fixed with 4% paraformaldehyde and acetylated with 0.25% acetic anhydride in 1.0M triethanolamine buffer. The sections were incubated for 2 hours at room temperature in pre-hybridization buffer, and were then hybridized with a probe concentration of 200μg per ml at 70° C, overnight in a humidified chamber. Following stringent washes in SSC buffers, the immunological detection steps were performed at room temperature. The alkaline phosphatase labeled, anti-digoxigenin antibody and detection buffers were purchased from Boehringer Mannheim. The sections were treated with a blocking buffer for one hour and incubated with antibody solution at a 1:5000 dilution for 2 hours, both at room temperature. Colored precipitate was formed by exposure to the alkaline phosphatase substrate nitrobluetetrazoliumchloride/ bromo-chloroindlylphosphate.

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