scholarly journals Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

2021 ◽  
Author(s):  
Kazuto Yoshimi ◽  
Kohei TAKESHITA ◽  
Noriyuki Kodera ◽  
Satomi Shibumura ◽  
Yuko Yamauchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Speed ◽  
Dna Helicase ◽  
Type I ◽  
Loop Formation ◽  
Force Microscopy ◽  
Atomic Force ◽  
Target Strand ◽  
Target Dna

Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific ssDNA cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target dsDNA substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.

MORPHOLOGY OF ADSORBED COLLAGEN LAYERS OBSERVED BY ATOMIC FORCE MICROSCOPY

2009 ◽  
Vol 21 (05) ◽  
pp. 311-316
Author(s):  
Yih-Pey Yang ◽  
Chia-Chi Lin
Keyword(s):  
Atomic Force Microscopy ◽  
Type I Collagen ◽  
Fibrillar Structure ◽  
Collagen Molecule ◽  
Nanometer Scale ◽  
Type I ◽  
Force Microscopy ◽  
Atomic Force

The interaction between cells and biomaterials strongly depends on the assembled structure of collagen adsorption upon the solid surface. Due to its self-assembling property, Type I collagen may aggregate and form fibrils in vivo and in vitro. This study utilizes an atomic force microscope to investigate nanometer-scale organization of adsorbed Type I collagen layers on mica and on poly(methyl methacrylate) (PMMA). We have observed various film morphologies, depending on substrate hydrophobicity and the state of collagen solution used. On mica, the atomic force microscopy (AFM) study obtains dense felt-like structures of randomly distributed assemblies. Images of network-like assemblies composed of interwoven fibrils appear on PMMA. According to the above results, we believe that these assemblies are associated at the interface rather than aggregated in the solution. This work also investigates the adsorbed collagen structure on PMMA after collagen aggregation in solution, to realize the relation between adsorption and aggregation. Consequently, the result exhibits a dendritic fibrillar structure adsorbed on PMMA, following collagen molecule aggregation, to form a fibrillar structure in the solution. This result suggests that the adsorption of aggregates preformed in the solution is preferable to collagen molecules adsorption. This research created all assembled structures of adsorbed collagen layers in nanometer-scale thickness.

scholarly journals Ultrastructure of influenza virus ribonucleoprotein complexes during viral RNA synthesis

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Masahiro Nakano ◽  
Yukihiko Sugita ◽  
Noriyuki Kodera ◽  
Sho Miyamoto ◽  
Yukiko Muramoto ◽  
...  
Keyword(s):  
Influenza A ◽  
High Speed ◽  
Rna Synthesis ◽  
Ultrastructural Feature ◽  
Helical Structures ◽  
Force Microscopy ◽  
Ribonucleoprotein Complexes ◽  
Atomic Force ◽  
Viral Genomic Rna

AbstractThe single-stranded, negative-sense, viral genomic RNA (vRNA) of influenza A virus is encapsidated by viral nucleoproteins (NPs) and an RNA polymerase to form a ribonucleoprotein complex (vRNP) with a helical, rod-shaped structure. The vRNP is responsible for transcription and replication of the vRNA. However, the vRNP conformation during RNA synthesis is not well understood. Here, using high-speed atomic force microscopy and cryo-electron microscopy, we investigated the native structure of influenza A vRNPs during RNA synthesis in vitro. Two distinct types of vRNPs were observed in association with newly synthesized RNAs: an intact, helical rod-shaped vRNP connected with a folded RNA and a deformed vRNP associated with a looped RNA. Interestingly, the looped RNA was a double-stranded RNA, which likely comprises a nascent RNA and the template RNA detached from NPs of the vRNP. These results suggest that while some vRNPs keep their helical structures during RNA synthesis, for the repeated cycle of RNA synthesis, others accidentally become structurally deformed, which likely results in failure to commence or continue RNA synthesis. Thus, our findings provide the ultrastructural feature of vRNPs during RNA synthesis.

scholarly journals Ultrastructure of influenza virus ribonucleoprotein complexes during viral RNA synthesis

2021 ◽  
Author(s):  
Masahiro Nakano ◽  
Yukihiko Sugita ◽  
Noriyuki Kodera ◽  
Sho Miyamoto ◽  
Yukiko Muramoto ◽  
...  
Keyword(s):  
Influenza A ◽  
High Speed ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Helical Structures ◽  
Force Microscopy ◽  
Ribonucleoprotein Complexes ◽  
Atomic Force ◽  
Transcription And Replication

Abstract The single-stranded, negative-sense, viral genomic RNA (vRNA) of influenza A virus is encapsidated by viral nucleoproteins and an RNA polymerase to form a ribonucleoprotein complex (vRNP) with a helical, rod-shaped structure. The vRNP is responsible for transcription and replication of the vRNA. However, the vRNP conformation during viral RNA synthesis is not well understood. Here, using high-speed atomic force microscopy and cryo-electron microscopy, we investigated the native structure of influenza A vRNPs during RNA synthesis in vitro. Two distinct types of vRNPs were observed in association with newly synthesized RNAs: an intact, helical rod-shaped vRNP connected with a structured viral RNA product and a deformed vRNP associated with a looped, double-stranded RNA, composed of a template vRNA and a nascent RNA. These results suggest that while some vRNPs keep their helical structures during viral RNA synthesis, probably for the repeated cycle of transcription and/or replication, others accidentally become structurally deformed, which probably results in failure to commence or continue RNA synthesis. Thus, our findings provide the ultrastructural basis of viral RNA synthesis and advance our knowledge of the mechanism of viral transcription and replication.

scholarly journals DNA looping by protamine follows a nonuniform spatial distribution

2021 ◽  
Author(s):  
Ryan B. McMillan ◽  
Victoria D. Kuntz ◽  
Luka M. Devenica ◽  
Hilary Bediako ◽  
Ashley R. Carter
Keyword(s):  
Spatial Distribution ◽  
Dna Origami ◽  
Dna Looping ◽  
Loop Formation ◽  
Force Microscopy ◽  
Atomic Force ◽  
Multivalent Cation ◽  
Structural Maintenance Of Chromosomes

ABSTRACTDNA looping plays an important role in cells in both regulating and protecting the genome. Often, studies of looping focus on looping by prokaryotic transcription factors like lac repressor or by structural maintenance of chromosomes (SMC) proteins such as condensin. Here, however, we are interested in a different looping method whereby multivalent cations (charge≥+3), such as protamine proteins, neutralize the DNA, causing it to form loops and toroids. We considered two previously proposed mechanisms for DNA looping by protamine. In the first mechanism, protamine stabilizes spontaneous DNA fluctuations, forming randomly distributed loops along the DNA. In the second mechanism, protamine binds and bends the DNA to form a loop, creating a distribution of loops that is biased by protamine binding. To differentiate between these mechanisms, we imaged both spontaneous and protamine-induced loops on short-length (≤ 1 μm) DNA fragments using atomic force microscopy (AFM). We then compared the spatial distribution of the loops to several model distributions. A random looping model, which describes the mechanism of spontaneous DNA folding, fit the distribution of spontaneous loops, but it did not fit the distribution of protamine-induced loops. Specifically, it overestimated the number of loops that form at the ends of the molecule and failed to predict a peak in the spatial distribution of loops at an intermediate location along the DNA. An electrostatic multibinding model, which was created to mimic the bind-and-bend mechanism of protamine, was a better fit of the distribution of protamine-induced loops. In this model, multiple protamines bind to the DNA electrostatically within a particular region along the DNA to coordinate the formation of a loop. We speculate that these findings will impact our understanding of protamine’s in vivo role for looping DNA into toroids and the mechanism of DNA condensation by multivalent cations more broadly.SIGNIFICANCEDNA looping is important in a variety of both in vivo functions (e.g. gene regulation) and in vitro applications (e.g. DNA origami). Here, we sought a mechanistic understanding of DNA looping by multivalent cations (≥+3), which condense DNA into loops and toroids. One such multivalent cation is the protein protamine, which condenses DNA in sperm. We investigated the mechanism for loop formation by protamine and found that the experimental data was consistent with an electrostatic multibinding model in which two protamines bind electrostatically to the DNA within a 50-nm region to form a loop. This model is likely general to all multivalent cations and may be helpful in applications involving toroid formation or DNA nanoengineering.

scholarly journals Sustainable Coated Nanostructures Based on Alginate and Electrospun Collagen Loaded with Antimicrobial Agents

Coatings ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 121
Author(s):  
Ecaterina Matei ◽  
Carmen Gaidau ◽  
Maria Râpă ◽  
Laura Mihaela Stefan ◽  
Lia-Mara Ditu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antimicrobial Agents ◽  
Biological Properties ◽  
Wound Dressings ◽  
Bacterial Strains ◽  
Force Microscopy ◽  
Atomic Force ◽  
Nanostructured Systems

In this study, sodium alginate film (Alg) was coated with electrospun collagen glue (Col) extracted from rabbit skin waste, loaded with different commercial antimicrobial agents (chitosan, AG425K and ZnONPs) and investigated in terms of morphological, structural and biological properties. The coated nanostructures were characterized using scanning electron microscopy coupled with the energy-dispersive X-ray (SEM/EDS), Attenuated Total Reflectance Fourier-Transform Infrared spectroscopy (ATR FT-IR), and Atomic Force Microscopy (AFM) tests. The cytotoxicity was investigated on murine L929 fibroblasts using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide salt (MTT) and lactate dehydrogenase (LDH) assays. Microbiological tests were performed against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Candida albicans ATCC 27853 standard strains. In vitro cell culture tests showed a good cytocompatibility of the coated nanostructured systems, except the sample loaded with ZnONPs, which exhibited a highly cytotoxic effect. Alg-Col-ZnONPs nanostructure inhibited the growth and multiplication of the Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 bacterial strains. The results of new coated nanostructures may be useful for the development of sustainable biomaterials in a circular economy, with bioactive properties for medical wound dressings.

scholarly journals Protamine loops DNA in multiple steps

2020 ◽  
Vol 48 (11) ◽  
pp. 6108-6119
Author(s):  
Obinna A Ukogu ◽  
Adam D Smith ◽  
Luka M Devenica ◽  
Hilary Bediako ◽  
Ryan B McMillan ◽  
...  
Keyword(s):  
Radius Of Curvature ◽  
Loop Current ◽  
Dna Molecules ◽  
Loop Formation ◽  
Step Process ◽  
Force Microscopy ◽  
Atomic Force ◽  
Dna Loop ◽  
One Step

Abstract Protamine proteins dramatically condense DNA in sperm to almost crystalline packing levels. Here, we measure the first step in the in vitro pathway, the folding of DNA into a single loop. Current models for DNA loop formation are one-step, all-or-nothing models with a looped state and an unlooped state. However, when we use a Tethered Particle Motion (TPM) assay to measure the dynamic, real-time looping of DNA by protamine, we observe the presence of multiple folded states that are long-lived (∼100 s) and reversible. In addition, we measure folding on DNA molecules that are too short to form loops. This suggests that protamine is using a multi-step process to loop the DNA rather than a one-step process. To visualize the DNA structures, we used an Atomic Force Microscopy (AFM) assay. We see that some folded DNA molecules are loops with a ∼10-nm radius and some of the folded molecules are partial loops—c-shapes or s-shapes—that have a radius of curvature of ∼10 nm. Further analysis of these structures suggest that protamine is bending the DNA to achieve this curvature rather than increasing the flexibility of the DNA. We therefore conclude that protamine loops DNA in multiple steps, bending it into a loop.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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