scholarly journals Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immuno-staining.

RNA ◽  
2021 ◽  
pp. rna.078895.121
Author(s):  
Prasath Paramasivam ◽  
Martin Stoter ◽  
Eloina Corradi ◽  
Irene Dalla Costa ◽  
Andreas Hoijer ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Single Molecule ◽  
Molecular Beacon ◽  
Cultured Cells ◽  
Endosomal Escape ◽  
Nucleic Acid Therapeutics ◽  
Optimized Protocol ◽  
Rna Probes ◽  
Cellular Compartments ◽  
Cell Fixation

Detection of nucleic acids within sub-cellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is particularly important for the delivery of nucleic acid therapeutics which depends on endocytic uptake and endosomal escape. However, the current protocols fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization and immuno-staining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels which escape detection using conventional procedures. The optimized protocol proved effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in-situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids in cultured cells.

scholarly journals Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immuno-staining

2021 ◽  
Author(s):  
Prasath Paramasivam ◽  
Martin Stoter ◽  
Eloina Corradi ◽  
Irene Dalla Costa ◽  
Andreas Hoijer ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Single Molecule ◽  
Molecular Beacon ◽  
Immunofluorescence Microscopy ◽  
Endosomal Escape ◽  
Nucleic Acid Therapeutics ◽  
Rna Probes ◽  
Cellular Compartments ◽  
Cell Fixation

Detection of nucleic acids within sub-cellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is important also for the delivery of nucleic acid therapeutics which depends on endocytic uptake and endosomal escape. However, the current methods fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization and immuno-staining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels which escape detection using conventional procedures. Additionally, we demonstrated the protocol to be effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in-situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids.

A Molecular Logic Gate Enables Super-Resolved Imaging of Intracellular Lipid Droplets

2019 ◽  
Author(s):  
Adam Eördögh ◽  
Carolina Paganini ◽  
Dorothea Pinotsi ◽  
Paolo Arosio ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Single Molecule ◽  
Lipid Droplets ◽  
Neutral Lipids ◽  
Logic Gate ◽  
Living Cells ◽  
Three Dimensions ◽  
Single Molecule Imaging ◽  
Molecular Logic Gate ◽  
Molecular Logic ◽  
Cellular Compartments

<div>Photoactivatable dyes enable single-molecule imaging in biology. Despite progress in the development of new fluorophores and labeling strategies, many cellular compartments remain difficult to image beyond the limit of diffraction in living cells. For example, lipid droplets, which are organelles that contain mostly neutral lipids, have eluded single-molecule imaging. To visualize these challenging subcellular targets, it is necessary to develop new fluorescent molecular devices beyond simple on/off switches. Here, we report a fluorogenic molecular logic gate that can be used to image single molecules associated with lipid droplets with excellent specificity. This probe requires the subsequent action of light, a lipophilic environment and a competent nucleophile to produce a fluorescent product. The combination of these requirements results in a probe that can be used to image the boundary of lipid droplets in three dimensions with resolutions beyond the limit of diffraction. Moreover, this probe enables single-molecule tracking of lipids within and between droplets in living cells.</div>

scholarly journals Encapsulation and Ultrasound-Triggered Release of G-Quadruplex DNA in Multilayer Hydrogel Microcapsules

Polymers ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 1342 ◽  
Author(s):  
Aaron Alford ◽  
Brenna Tucker ◽  
Veronika Kozlovskaya ◽  
Jun Chen ◽  
Nirzari Gupta ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Defense Mechanisms ◽  
The Body ◽  
Stimuli Responsive ◽  
Triggered Release ◽  
Quadruplex Dna ◽  
Nucleic Acid Therapeutics ◽  
Wide Range ◽  
G Quadruplex ◽  
Hydrogel Capsules

Nucleic acid therapeutics have the potential to be the most effective disease treatment strategy due to their intrinsic precision and selectivity for coding highly specific biological processes. However, freely administered nucleic acids of any type are quickly destroyed or rendered inert by a host of defense mechanisms in the body. In this work, we address the challenge of using nucleic acids as drugs by preparing stimuli responsive poly(methacrylic acid)/poly(N-vinylpyrrolidone) (PMAA/PVPON)n multilayer hydrogel capsules loaded with ~7 kDa G-quadruplex DNA. The capsules are shown to release their DNA cargo on demand in response to both enzymatic and ultrasound (US)-triggered degradation. The unique structure adopted by the G-quadruplex is essential to its biological function and we show that the controlled release from the microcapsules preserves the basket conformation of the oligonucleotide used in our studies. We also show that the (PMAA/PVPON) multilayer hydrogel capsules can encapsulate and release ~450 kDa double stranded DNA. The encapsulation and release approaches for both oligonucleotides in multilayer hydrogel microcapsules developed here can be applied to create methodologies for new therapeutic strategies involving the controlled delivery of sensitive biomolecules. Our study provides a promising methodology for the design of effective carriers for DNA vaccines and medicines for a wide range of immunotherapies, cancer therapy and/or tissue regeneration therapies in the future.

scholarly journals Preparation of Mycobacterium smegmatis porin A (MspA) nanopores for single molecule sensing of nucleic acids

2021 ◽  
Vol 7 (5) ◽  
pp. 355-364
Author(s):  
Wang Yuqin ◽  
◽  
Fan Pingping ◽  
Zhang Shanyu ◽  
Yan Shuanghong ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Single Molecule ◽  
Mycobacterium Smegmatis

scholarly journals An optimized live bacterial delivery platform for the production and delivery of therapeutic nucleic acids and proteins

2021 ◽  
Author(s):  
Darcy S.O. Mora ◽  
Madeline Cox ◽  
Forgivemore Magunda ◽  
Ashley B. Williams ◽  
Lyndsey Linke
Keyword(s):  
Epithelial Cells ◽  
Mouse Model ◽  
Nucleic Acids ◽  
Endosomal Escape ◽  
Therapeutic Window ◽  
Target Cells ◽  
Full Potential ◽  
Wide Range ◽  
Delivery Platform

There is an unmet need for delivery platforms that realize the full potential of next-generation therapeutic and vaccine technologies, especially those that require intracellular delivery of nucleic acids. The in vivo usefulness of the current state-of-the-art delivery systems is limited by numerous intrinsic weaknesses, including lack of targeting specificity, inefficient entry and endosomal escape into target cells, undesirable immune activation, off-target effects, a small therapeutic window, limited genetic encoding and cargo capacity, and manufacturing challenges. Here we present our characterization of a delivery platform based on the use of engineered live, tissue-targeting, non-pathogenic bacteria (Escherichia coli strain SVC1) for intracellular cargo delivery. The SVC1 bacteria are engineered to specifically bind to epithelial cells via a surface-expressed targeting ligand, to escape the endosome upon intracellularization, and to have minimal immunogenicity. Here we report findings on key features of this system. First, we demonstrated that bacterial delivery of a short hairpin RNA (shRNA) can target and silence a gene in an in vitro mammalian respiratory cell model. Next, we used an in vivo mouse model to demonstrate that SVC1 bacteria are invasive to epithelial cells of various tissues and organs (eye, nose, mouth, stomach, vagina, skeletal muscle, and lungs) via local administration. We also showed that repeat dosing of SVC1 bacteria to the lungs is minimally immunogenic and that it does not have adverse effects on tissue homeostasis. Finally, to validate the potential of SVC1 bacteria in therapeutic applications, we demonstrated that bacterial delivery of influenza- targeting shRNAs to the respiratory tissues can mitigate viral replication in a mouse model of influenza infection. Our ongoing work is focused on further refining this platform for efficient delivery of nucleic acids, gene editing machinery, and therapeutic proteins, and we expect that this platform technology will enable a wide range of advanced therapeutic approaches.

scholarly journals Methods for protein delivery into cells: from current approaches to future perspectives

2020 ◽  
Vol 48 (2) ◽  
pp. 357-365
Author(s):  
Chalmers Chau ◽  
Paolo Actis ◽  
Eric Hewitt
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Protein Delivery ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Cellular Damage ◽  
Future Perspectives ◽  
Chemically Modified ◽  
Recent Developments ◽  
Direct Delivery

The manipulation of cultured mammalian cells by the delivery of exogenous macromolecules is one of the cornerstones of experimental cell biology. Although the transfection of cells with DNA expressions constructs that encode proteins is routine and simple to perform, the direct delivery of proteins into cells has many advantages. For example, proteins can be chemically modified, assembled into defined complexes and subject to biophysical analyses prior to their delivery into cells. Here, we review new approaches to the injection and electroporation of proteins into cultured cells. In particular, we focus on how recent developments in nanoscale injection probes and localized electroporation devices enable proteins to be delivered whilst minimizing cellular damage. Moreover, we discuss how nanopore sensing may ultimately enable the quantification of protein delivery at single-molecule resolution.

A facile deoxyuridine/biotin-modified molecular beacon for simultaneous detection of proteins and nucleic acids via a label-free and background-eliminated fluorescence assay

The Analyst ◽  
2019 ◽  
Vol 144 (18) ◽  
pp. 5504-5510 ◽  
Author(s):  
Fei Yin ◽  
Liqi Liu ◽  
Xia Sun ◽  
Laiyong Hou ◽  
Yu Lu ◽  
...  
Keyword(s):  
Early Diagnosis ◽  
Nucleic Acids ◽  
Molecular Beacon ◽  
Simultaneous Detection ◽  
Cancer Biomarkers ◽  
Label Free ◽  
Early Diagnosis Of Cancer ◽  
Different Types ◽  
Diagnosis Of Cancer ◽  
Modified Molecular Beacon

Simultaneous detection of different types of cancer biomarkers (nucleic acids and proteins) could facilitate early diagnosis of cancer and clinical treatment.

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