Direct visualization of both DNA and RNA quadruplexes in human cells via an uncommon spectroscopic method

ABSTRACTCRISPR technology has opened up many diverse genome editing possibilities in human somatic cells, but has been limited in the therapeutic realm by both potential off-target effects and low genome modification efficiencies. Recent advancements to combat these limitations include delivering Cas9 nucleases directly to cells as highly purified ribonucleoproteins (RNPs) instead of the conventional plasmid DNA and RNA-based approaches. Here, we extend RNP-based delivery in cell culture to a less characterized CRISPR format which implements paired Cas9 nickases. The use of paired nickase Cas9 RNP system, combined with a GMP-compliant non-viral delivery technology, enables editing in human cells with high specificity and high efficiency, a development that opens up the technology for further exploration into a more therapeutic role.

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Inhibition by selenium of DNA and RNA synthesis in normal and malignant human cells in vitro

Cancer Letters ◽

10.1016/0304-3835(92)90211-d ◽

1992 ◽

Vol 65 (1) ◽

pp. 43-49 ◽

Cited By ~ 14

Author(s):

Fikrat I. Abdullaev ◽

Christina MacVicar ◽

Gerald D. Frenkel

Keyword(s):

Rna Synthesis ◽

Human Cells ◽

Dna And Rna ◽

Dna And Rna Synthesis

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DNA and RNA editing without sequence limitation using the flap endonuclease 1 guided by hairpin DNA probes

Nucleic Acids Research ◽

10.1093/nar/gkaa843 ◽

2020 ◽

Vol 48 (20) ◽

pp. e117-e117

Author(s):

Kun Tian ◽

Yongjian Guo ◽

Bingjie Zou ◽

Liang Wang ◽

Yun Zhang ◽

...

Keyword(s):

Rna Editing ◽

Human Cells ◽

Hairpin Dna ◽

Flap Endonuclease 1 ◽

Dna And Rna ◽

Rna Targets ◽

Single Base Mismatch ◽

Compact Size

Abstract Here, we characterized a flap endonuclease 1 (FEN1) plus hairpin DNA probe (hpDNA) system, designated the HpSGN system, for both DNA and RNA editing without sequence limitation. The compact size of the HpSGN system make it an ideal candidate for in vivo delivery applications. In vitro biochemical studies showed that the HpSGN system required less nuclease to cleave ssDNA substrates than the SGN system we reported previously by a factor of ∼40. Also, we proved that the HpSGN system can efficiently cleave different RNA targets in vitro. The HpSGN system cleaved genomic DNA at an efficiency of ∼40% and ∼20% in bacterial and human cells, respectively, and knocked down specific mRNAs in human cells at a level of ∼25%. Furthermore, the HpSGN system was sensitive to the single base mismatch at the position next to the hairpin both in vitro and in vivo. Collectively, this study demonstrated the potential of developing the HpSGN system as a small, effective, and specific editing tool for manipulating both DNA and RNA without sequence limitation.

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The first successful observation of in-cell NMR signals of DNA and RNA in living human cells

Physical Chemistry Chemical Physics ◽

10.1039/c7cp05188c ◽

2018 ◽

Vol 20 (5) ◽

pp. 2982-2985 ◽

Cited By ~ 18

Author(s):

Yudai Yamaoki ◽

Ayaka Kiyoishi ◽

Masayuki Miyake ◽

Fumi Kano ◽

Masayuki Murata ◽

...

Keyword(s):

Pore Formation ◽

Human Cells ◽

Dna And Rna ◽

Nmr Signals ◽

Successful Observation

The first observation of NMR signals of DNA/RNA introduced into living human cells by means of pore formation by SLO and resealing.

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Direct visualization of the dynamics of antigen presentation in human cells infected with cytomegalovirus revealed by antibodies mimicking TCR specificity

European Journal of Immunology ◽

10.1002/eji.200939875 ◽

2010 ◽

Vol 40 (6) ◽

pp. 1552-1565 ◽

Cited By ~ 24

Author(s):

Oryan Makler ◽

Kfir Oved ◽

Nir Netzer ◽

Dana Wolf ◽

Yoram Reiter

Keyword(s):

Antigen Presentation ◽

Human Cells ◽

Direct Visualization

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Direct visualization of phase-separated domains in lipid membranes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100082315 ◽

1978 ◽

Vol 36 (2) ◽

pp. 290-291

Author(s):

S. W. Hui ◽

T. P. Stewart

Keyword(s):

Lipid Membranes ◽

Structural Information ◽

Freeze Fracture ◽

Biological Molecules ◽

Vacuum Drying ◽

Direct Visualization ◽

X Ray Diffraction ◽

Electron Microscopic ◽

X Ray ◽

Hydrocarbon Chains

Direct electron microscopic study of biological molecules has been hampered by such factors as radiation damage, lack of contrast and vacuum drying. In certain cases, however, the difficulties may be overcome by using redundent structural information from repeating units and by various specimen preservation methods. With bilayers of phospholipids in which both the solid and fluid phases co-exist, the ordering of the hydrocarbon chains may be utilized to form diffraction contrast images. Domains of different molecular packings may be recgnizable by placing properly chosen filters in the diffraction plane. These domains would correspond to those observed by freeze fracture, if certain distinctive undulating patterns are associated with certain molecular packing, as suggested by X-ray diffraction studies. By using an environmental stage, we were able to directly observe these domains in bilayers of mixed phospholipids at various temperatures at which their phases change from misible to inmissible states.

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High-resolution nucleic acid sequence mapping via in situ hybridization at the Electron Microscope level

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140130 ◽

1995 ◽

Vol 53 ◽

pp. 752-753

Author(s):

B.A. Hamkalo ◽

S. Narayanswami ◽

A.P. Kausch

Keyword(s):

Nucleic Acid ◽

Interphase Nucleus ◽

Genome Mapping ◽

Precise Location ◽

Electron Microscope Level ◽

Rna Sequences ◽

Fundamental Interest ◽

Whole Cells ◽

Dna And Rna

The availability of nonradioactive methods to label nucleic acids an the resultant rapid and greater sensitivity of detection has catapulted the technique of in situ hybridization to become the method of choice to locate of specific DNA and RNA sequences on chromosomes and in whole cells in cytological preparations in many areas of biology. It is being applied to problems of fundamental interest to basic cell and molecular biologists such as the organization of the interphase nucleus in the context of putative functional domains; it is making major contributions to genome mapping efforts; and it is being applied to the analysis of clinical specimens. Although fluorescence detection of nucleic acid hybrids is routinely used, certain questions require greater resolution. For example, very closely linked sequences may not be separable using fluorescence; the precise location of sequences with respect to chromosome structures may be below the resolution of light microscopy(LM); and the relative positions of sequences on very small chromosomes may not be feasible.

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Scanning tunneling microscopy (STM) of DNA and RNA

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100152148 ◽

1989 ◽

Vol 47 ◽

pp. 34-35

Author(s):

Patricia G. Arscott ◽

Gil Lee ◽

Victor A. Bloomfield ◽

D. Fennell Evans

Keyword(s):

Molecular Structures ◽

Inorganic Materials ◽

Resolving Power ◽

Scanning Tunneling ◽

Tunneling Microscopy ◽

Form And Function ◽

Dna And Rna ◽

Calf Thymus ◽

And Function ◽

The Relationship

STM is one of the most promising techniques available for visualizing the fine details of biomolecular structure. It has been used to map the surface topography of inorganic materials in atomic dimensions, and thus has the resolving power not only to determine the conformation of small molecules but to distinguish site-specific features within a molecule. That level of detail is of critical importance in understanding the relationship between form and function in biological systems. The size, shape, and accessibility of molecular structures can be determined much more accurately by STM than by electron microscopy since no staining, shadowing or labeling with heavy metals is required, and there is no exposure to damaging radiation by electrons. Crystallography and most other physical techniques do not give information about individual molecules.We have obtained striking images of DNA and RNA, using calf thymus DNA and two synthetic polynucleotides, poly(dG-me5dC)·poly(dG-me5dC) and poly(rA)·poly(rU).

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