scholarly journals Population scale nucleic acid delivery to Caenorhabditis elegans via electroporation

2020 ◽  
Author(s):  
Anastasia S. Khodakova ◽  
Daniela Vidal Vilchis ◽  
Ferdinand Amanor ◽  
Buck S. Samuel
Keyword(s):  
Nucleic Acid ◽  
Prolonged Exposure ◽  
Nucleic Acid Delivery ◽  
Guide Rna ◽  
E Coli ◽  
Genetic Manipulations ◽  
Free Living Nematode ◽  
Population Scale ◽  
Nucleic Acids Delivery ◽  
Genetic Targeting

ABSTRACTThe free-living nematode C.elegans remains one of the most robust and flexible genetic systems for inter-rogating the complexities of animal biology. Targeted genetic manipulations, such as RNA interference (RNAi), CRISPR/Cas9- or array-based transgenesis, all depend on initial delivery of nucleic acids. Delivery of dsRNA by feeding can be effective, but expression in E. coli is not conducive to experiments intended to remain sterile or with defined microbial communities. Soaking-based delivery requires prolonged exposure of animals to high material concentrations without a food source and is of limited throughput. Last, microinjection of individual animals can precisely deliver materials to animals’ germlines, but is limited by the need to target and inject each animal one-by-one. Thus, we sought to address some of these challenges in nucleic acid delivery by developing a population-scale delivery method. We demonstrate efficient electroporation-mediated delivery of dsRNA throughout the worm and effective RNAi-based silencing, including in the germline. Finally, we show that guide RNA delivered by electroporation can be utilized by transgenic Cas9 expressing worms for population-scale genetic targeting. Together, these methods expand the scale and scope of genetic methodologies that can be applied to the C.elegans system.

scholarly journals Population scale nucleic acid delivery to Caenorhabditis elegans via electroporation

2021 ◽  
Author(s):  
Anastasia S Khodakova ◽  
Daniela Vidal Vilchis ◽  
Dana Blackburn ◽  
Ferdinand Amanor ◽  
Buck S Samuel
Keyword(s):  
Nucleic Acid ◽  
Prolonged Exposure ◽  
Nucleic Acid Delivery ◽  
Guide Rna ◽  
E Coli ◽  
Genetic Manipulations ◽  
Free Living Nematode ◽  
Population Scale ◽  
Nucleic Acids Delivery ◽  
Genetic Targeting

Abstract The free-living nematode C.elegans remains one of the most robust and flexible genetic systems for interrogating the complexities of animal biology. Targeted genetic manipulations, such as RNA interference (RNAi), CRISPR/Cas9- or array-based transgenesis, all depend on initial delivery of nucleic acids. Delivery of dsRNA by feeding can be effective, but expression in E. coli is not conducive to experiments intended to remain sterile or with defined microbial communities. Soaking-based delivery requires prolonged exposure of animals to high material concentrations without a food source and is of limited throughput. Last, microinjection of individual animals can precisely deliver materials to animals’ germlines, but is limited by the need to target and inject each animal one-by-one. Thus, we sought to address some of these challenges in nucleic acid delivery by developing a population-scale delivery method. We demonstrate efficient electroporation-mediated delivery of dsRNA throughout the worm and effective RNAi-based silencing, including in the germline. Finally, we show that guide RNA delivered by electroporation can be utilized by transgenic Cas9 expressing worms for population-scale genetic targeting. Together, these methods expand the scale and scope of genetic methodologies that can be applied to the C.elegans system.

Non-Viral Nucleic Acid Delivery: Key Challenges and Future Directions

2011 ◽  
Vol 8 (3) ◽  
pp. 235-244 ◽  
Author(s):  
Mahmoud Elsabahy ◽  
Adil Nazarali ◽  
Marianna Foldvari
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Delivery ◽  
Viral Nucleic Acid ◽  
Future Directions

Charge‐Conversion Strategies for Nucleic Acid Delivery

2021 ◽  
pp. 2011103
Author(s):  
Kingshuk Dutta ◽  
Ritam Das ◽  
Jewel Medeiros ◽  
Pintu Kanjilal ◽  
S. Thayumanavan
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Delivery

scholarly journals Synthetic Histidine-Rich Peptides Inhibit Candida Species and Other Fungi In Vitro: Role of Endocytosis and Treatment Implications

2006 ◽  
Vol 50 (8) ◽  
pp. 2797-2805 ◽  
Author(s):  
Jingsong Zhu ◽  
Paul W. Luther ◽  
Qixin Leng ◽  
A. James Mixson
Keyword(s):  
Nucleic Acid ◽  
Antifungal Activity ◽  
Antifungal Agents ◽  
Fungal Growth ◽  
Nucleic Acid Delivery ◽  
Histatin 5 ◽  
Ph Buffering ◽  
Endosomal Acidification

ABSTRACT A family of histidine-rich peptides, histatins, is secreted by the parotid gland in mammals and exhibits marked inhibitory activity against a number of Candida species. We were particularly interested in the mechanism by which histidine-rich peptides inhibit fungal growth, because our laboratory has synthesized a variety of such peptides for drug and nucleic acid delivery. In contrast to naturally occurring peptides that are linear, peptides made on synthesizers can be varied with respect to their degrees of branching. Using this technology, we explored whether histidine-lysine (HK) polymers of different complexities and degrees of branching affect the growth of several species of Candida. Polymers with higher degrees of branching were progressively more effective against Candida albicans, with the four-branched polymer, H2K4b, most effective. Furthermore, H2K4b accumulated efficiently in C. albicans, which may indicate its ability to transport other antifungal agents intracellularly. Although H2K4b had greater antifungal activity than histatin 5, their mechanisms were similar. Toxicity in C. albicans induced by histatin 5 or branched HK peptides was markedly reduced by 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate, an inhibitor of anion channels. We also determined that bafilomycin A1, an inhibitor of endosomal acidification, significantly decreased the antifungal activity of H2K4b. This suggests that the pH-buffering and subsequent endosomal-disrupting properties of histidine-rich peptides have a role in their antifungal activity. Moreover, the ability of the histidine component of these peptides to disrupt endosomes, which allows their escape from the lysosomal pathway, may explain why these peptides are both effective antifungal agents and nucleic acid delivery carriers.

Nucleic acid delivery using magnetic nanoparticles: the Magnetofection™ technology

2011 ◽  
Vol 2 (4) ◽  
pp. 471-482 ◽  
Author(s):  
Nicolas Laurent ◽  
Cédric Sapet ◽  
Loic Le Gourrierec ◽  
Elodie Bertosio ◽  
Olivier Zelphati
Keyword(s):  
Magnetic Nanoparticles ◽  
Nucleic Acid ◽  
Nucleic Acid Delivery

Arginine-rich hydrophobic polyethylenimine: Potent agent with simple components for nucleic acid delivery

2013 ◽  
Vol 60 ◽  
pp. 18-27 ◽  
Author(s):  
Hamideh Parhiz ◽  
Maryam Hashemi ◽  
Arash Hatefi ◽  
Wayne Thomas Shier ◽  
Sara Amel Farzad ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Delivery

Nucleic Acid Delivery with Chitosan Hydroxybenzotriazole

2010 ◽  
Vol 20 (3) ◽  
pp. 127-136 ◽  
Author(s):  
Praneet Opanasopit ◽  
Sunee Techaarpornkul ◽  
Theerasak Rojanarata ◽  
Tanasait Ngawhirunpat ◽  
Uracha Ruktanonchai
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Delivery

IMMU-53. STING-ING GLIOBLASTOMA WITH SPHERICAL NUCLEIC ACIDS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi104-vi105
Author(s):  
Akanksha Mahajan ◽  
Lisa Hurley ◽  
Serena Tommasini-Ghelfi ◽  
Corey Dussold ◽  
Alexander Stegh ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Surface ◽  
Biological Properties ◽  
Innate Immune ◽  
Limiting Factors ◽  
Nucleic Acid Delivery ◽  
Sting Pathway ◽  
Spherical Nucleic Acids

Abstract The Stimulator of Interferon Genes (STING) pathway represents a major innate immune sensing mechanism for tumor-derived DNA. Modified cyclic dinucleotides (CDNs) that mimic the endogenous STING ligand cGAMP are currently being explored in patients with solid tumors that are amenable to intratumoral delivery. Inadequate bioavailability and insufficient lipophilicity are limiting factors for clinical CDN development, in particular when consideration is given to systemic administration approaches. We have shown that the formulation of oligonucleotides into Spherical Nucleic Acid (SNA) nanostructures, i.e.,the presentation of oligonucleotides at high density on the surface of nanoparticle cores, lead to biochemical and biological properties that are radically different from those of linear oligonucleotides. First-generation brain-penetrant siRNA-based SNAs (NCT03020017, recurrent GBM) have recently completed early clinical trials. Here, we report the development of a STING-agonistic immunotherapy by targeting cGAS, the sensor of cytosolic dsDNA upstream of STING, with SNAs presenting dsDNA at high surface density. The strategy of using SNAs exploits the ability of cGAS to raise STING responses by delivering dsDNA and inducing the catalytic production of endogenous CDNs. SNA nanostructures carrying a 45bp IFN-simulating dsDNA oligonucleotide, the most commonly used and widely characterized cGAS activator, potently activated the cGAS-STING pathway in vitro and in vivo. In a poorly immunogenic and highly aggressive syngeneic mouse glioma model, in which tumours were well-established, only one dose of intranasal treatment with STING-SNAs decelerated tumour growth, improved survival and importantly, was well-tolerated. Our use of SNAs addresses the challenges of nucleic acid delivery to intracranial tumor sites via intranasal route, exploits the binding of dsDNA molecules on the SNA surface to enhance the formation of a dimeric cGAS:DNA complex and establishes cGAS-agonistic SNAs as a novel class of immune-stimulatory modalities for triggering innate immune responses against tumor.

scholarly journals Conditional guide RNA through two intermediate hairpins for programmable CRISPR/Cas9 function: building regulatory connections between endogenous RNA expressions

2020 ◽  
Vol 48 (20) ◽  
pp. 11773-11784
Author(s):  
Jiao Lin ◽  
Yan Liu ◽  
Peidong Lai ◽  
Huixia Ye ◽  
Liang Xu
Keyword(s):  
Nucleic Acid ◽  
Genetic Regulation ◽  
Binding Activity ◽  
Strand Displacement ◽  
Genetic Circuits ◽  
Reporter System ◽  
Gene Expressions ◽  
Guide Rna ◽  
Naturally Occurring ◽  
Novel Approach

Abstract A variety of nanodevices developed for nucleic acid computation provide great opportunities to construct versatile synthetic circuits for manipulation of gene expressions. In our study, by employing a two-hairpin mediated nucleic acid strand displacement as a processing joint for conditional guide RNA, we aim to build artificial connections between naturally occurring RNA expressions through programmable CRISPR/Cas9 function. This two-hairpin joint possesses a sequence-switching machinery, in which a random trigger strand can be processed to release an unconstrained sequence-independent strand and consequently activate the self-inhibitory guide RNA for conditional gene regulation. This intermediate processor was characterized by the fluorescence reporter system and applied for regulation of the CRISPR/Cas9 binding activity. Using plasmids to generate this sequence-switching machinery in situ, we achieved the autonomous genetic regulation of endogenous RNA expressions controlled by other unrelated endogenous RNAs in both E. coli and human cells. Unlike previously reported strand-displacement genetic circuits, this advanced nucleic acid nanomachine provides a novel approach that can establish regulatory connections between naturally occurring endogenous RNAs. In addition to CRISPR systems, we anticipate this two-hairpin machine can serve as a general processing joint for wide applications in the development of other RNA-based genetic circuits.

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