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1.
Nat Chem Biol ; 13(11): 1187-1194, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28945233

ABSTRACT

Quantitative measurement of transcription rates in live cells is important for revealing mechanisms of transcriptional regulation. This is particularly challenging when measuring the activity of RNA polymerase III (Pol III), which transcribes growth-promoting small RNAs. To address this issue, we developed Corn, a genetically encoded fluorescent RNA reporter suitable for quantifying RNA transcription in cells. Corn binds and induces fluorescence of 3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime, which resembles the fluorophore found in red fluorescent protein (RFP). Notably, Corn shows high photostability, enabling quantitative fluorescence imaging of mTOR-dependent Pol III transcription. We found that, unlike actinomycin D, mTOR inhibitors resulted in heterogeneous transcription suppression in individual cells. Quantitative imaging of Corn-tagged Pol III transcript levels revealed distinct Pol III transcription 'trajectories' elicited by mTOR inhibition. Together, these studies provide an approach for quantitative measurement of Pol III transcription by direct imaging of Pol III transcripts containing a photostable RNA-fluorophore complex.


Subject(s)
Aptamers, Nucleotide/genetics , Chromophore-Assisted Light Inactivation , Fluorescent Dyes/metabolism , Optical Imaging/methods , RNA Polymerase III/analysis , Transcription, Genetic , Aptamers, Nucleotide/metabolism , Base Pairing , Base Sequence , Gene Expression Regulation , HEK293 Cells , Humans , Luminescent Proteins/metabolism , Nucleic Acid Conformation , RNA Polymerase III/genetics , Sirolimus/analogs & derivatives , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Red Fluorescent Protein
2.
Chembiochem ; 18(18): 1814-1818, 2017 09 19.
Article in English | MEDLINE | ID: mdl-28704573

ABSTRACT

Cyanine (Cy) dyes show a general propensity to localize in polarized mitochondria. This mitochondriotropism was used to perform a copper-free click reaction in the mitochondria of living cells. The in organello reaction of dyes Cy3 and Cy5 led to a product that was easily traceable by Förster resonance energy transfer (FRET). As determined by confocal laser scanning microscopy, the Cy3-Cy5 conjugate showed enhanced retention in mitochondria, relative to that of the starting compounds. This enhancement of a favorable property can be achieved by synthesis in organello, but not outside mitochondria.


Subject(s)
Carbocyanines/metabolism , Fluorescent Dyes/metabolism , Mitochondria/metabolism , Animals , Carbocyanines/chemistry , Cell Line , Click Chemistry , Copper/chemistry , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemistry , Microscopy, Confocal , Rats
3.
Nucleic Acids Res ; 43(9): 4650-60, 2015 May 19.
Article in English | MEDLINE | ID: mdl-25870407

ABSTRACT

Intracellular distribution of siRNA after in vitro transfection typically depends on lipopolyplexes, which must release the siRNA into the cytosol. Here, the fate of siRNAs was monitored by FRET-based live cell imaging. Subsequent to in situ observation of uptake and release processes, this approach allowed the observation of a number of hitherto uncharacterized intracellular distribution and degradation processes, commencing with a burst of endosomal releases, followed, in some cases, by fast siRNA influx into the nucleus. The continued observation of intact siRNA against a background of free fluorophores resulting from advanced degradation was possible by a specifically developed imaging algorithm, which identified populations of intact siRNA in pixels based on FRET. This proved to be essential in the end point definition of siRNA distribution, which typically featured partially degraded siRNA pools in perinuclear structures. Our results depict the initial 4 h as a critical time window, characterized by fast initial burst release into the cytosol, which lay the foundations for subsequent intracellular distribution of siRNA. Combination with a subsequent slower, but sustained release from endosomal reservoirs may contribute to the efficiency and duration of RNAi, and explain the success of lipopolyplexes in RNAi experiments in cell culture.


Subject(s)
RNA, Small Interfering/metabolism , Animals , Cell Line , Cell Nucleus/metabolism , Endosomes/metabolism , Fluorescence Resonance Energy Transfer , Microscopy, Confocal , RNA Transport , Rats , Transfection
4.
Pharm Res ; 32(6): 1957-74, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25488263

ABSTRACT

PURPOSE: Release of siRNA from nanoscale polyplexes is a crucial yet little investigated process, important during all stages of therapeutic research. Here we develop new methods to characterize polyplex stability early on in the development of new materials. METHODS: We used double fluorescent labeled siRNA to compare binding and stability of a panel of chemically highly diverse nanoscale polyplexes, including peptides, lipids, nanohydrogels, poly-L-lysine brushes, HPMA block copolymers and manganese oxide particles. Conventional EMSA and heparin competition methods were contrasted with a newly developed microscale thermophoresis (MST) assay, a near-equilibrium method that allows free choice of buffer conditions. Integrity of FRET-labeled siRNA was monitored in the presence of nucleases, in cell culture medium and inside living cells. This approach characterizes all relevant steps from polyplex stability, over uptake to in vitro knockdown capability. RESULTS: Diverging polyplex binding properties revealed drawbacks of conventional EMSA and heparin competition assays, where MST and FRET-based siRNA integrity measurements offered a better discrimination of differential binding strength. Since cell culture medium left siRNA in all polyplexes essentially intact, the relevant degradation events could be pinpointed to occur inside cells. Differential binding strength of the variegated polyplexes correlated only partially with intracellular degradation. The most successful compounds in RNAi showed intermediate binding strength in our assays. CONCLUSIONS: We introduce new methods for the efficient and informative characterization of siRNA polyplexes with special attention to stability. Comparing FRET-labeled siRNA in different polyplexes associates successful knockdown with intermediate siRNA stability in various steps from formulation to intracellular persistence.


Subject(s)
Nanoparticles , Nanotechnology/methods , RNA Interference , RNA, Small Interfering/metabolism , Transfection/methods , Biological Transport , Electrophoretic Mobility Shift Assay , Fluorescence Resonance Energy Transfer , Gene Expression Regulation , Gene Knockdown Techniques , Genes, Reporter , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , HeLa Cells , Humans , Kinetics , RNA Stability , RNA, Small Interfering/chemistry
5.
Biomacromolecules ; 15(7): 2440-8, 2014 Jul 14.
Article in English | MEDLINE | ID: mdl-24805163

ABSTRACT

Aiming at controlled modification of liposomal surface structures, we describe a postpreparational approach for surface derivatization of a new type of multifunctional, sterically stabilized liposomes. Application of dual centrifugation (DC) resulted in high encapsulation efficiencies above 50% at very small batch sizes with a total volume of 150 µL, which were conductive to fast and efficient optimization of variegated surface modification reactions. Cholesterol-polymer amphiphiles, including complex hyperbranched polyether structures bearing 1-4 terminal alkynes, were used in DC formulations to provide steric stabilization. The alkyne moieties were explored as anchors for the conjugation of small molecules to the liposomal surface via click chemistry, binding 350-450 fluorophores per liposome as examples for surface active molecules. Using Förster resonance energy transfer (FRET) spectroscopy, the conjugation reaction as well as the uptake of FRET-labeled liposomes by RBE4 cells was monitored, and the distribution of the fluorescent lipids among cellular structures and membranes could be studied. Thus, the combination of clickable hyperbranched amphiphiles and dual centrifugation provides access to well-defined liposomal formulations with a variety of surface moieties.


Subject(s)
Doxorubicin/analogs & derivatives , Polymers/pharmacology , Alkynes/chemistry , Animals , Brain/cytology , Brain/drug effects , Brain/metabolism , Cell Line , Click Chemistry , Doxorubicin/chemistry , Doxorubicin/pharmacology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Fluorescence Resonance Energy Transfer , Liposomes , Microscopy, Confocal , Polyethylene Glycols/chemistry , Polyethylene Glycols/pharmacology , Polymers/chemistry , Rats
6.
Biol Chem ; 393(1-2): 23-35, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22628296

ABSTRACT

Investigations into the fate of small interfering RNA (siRNA) after transfection may unravel new ways to improve RNA interference (RNAi) efficiency. Because intracellular degradation of RNA may prevent reliable observation of fluorescence-labeled siRNA, new tools for fluorescence microscopy are warranted to cover the considerable duration of the RNAi effect. Here, the characterization and application of new fluorescence resonance energy transfer (FRET) dye pairs for sensing the integrity of duplex siRNA is reported, which allows an assessment of the degradation status of an siRNA cell population by live cell imaging. A panel of high-yield fluorescent dyes has been investigated for their suitability as FRET pairs for the investigation of RNA inside the cell. Nine dyes in 13 FRET pairs were evaluated based on the performance in assays of photostability, cross-excitation, bleed-through, as well as on quantified changes of fluorescence as a consequence of, e.g., RNA strand hybridization and pH variation. The Atto488/Atto590 FRET pair has been applied to live cell imaging, and has revealed first aspects of unusual trafficking of intact siRNA. A time-lapse study showed highly dynamic movement of siRNA in large perinuclear structures. These and the resulting optimized FRET labeled siRNA are expected to have significant impact on future observations of labeled RNAs in living cells.


Subject(s)
Brain/cytology , Endothelial Cells/cytology , Fluorescent Dyes/analysis , RNA, Small Interfering/analysis , Animals , Brain/metabolism , Cell Survival , Cells, Cultured , Endothelial Cells/metabolism , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemistry , Hydrogen-Ion Concentration , Microscopy, Confocal , RNA, Small Interfering/chemistry , Rats
7.
Chem Commun (Camb) ; 48(89): 11014-6, 2012 Nov 18.
Article in English | MEDLINE | ID: mdl-23037931

ABSTRACT

A propargyl containing guanosine phosphoramidite was synthesized and incorporated into siRNA, enabling click-ligation with an azido fluorophore onto the nucleobase sugar edge. Duplex stability was not affected by labeling at this new site, which allowed deconvolution of the effects of label, structure and attachment site on RNAi activity.


Subject(s)
Carbohydrates/chemistry , Guanosine/chemistry , Organophosphorus Compounds/chemistry , RNA, Small Interfering/chemistry , RNA/chemistry , Click Chemistry , Guanosine/chemical synthesis , Molecular Structure , Organophosphorus Compounds/chemical synthesis
8.
ACS Nano ; 6(3): 2198-214, 2012 Mar 27.
Article in English | MEDLINE | ID: mdl-22381078

ABSTRACT

Oligonucleotides such as short, double-stranded RNA (siRNA) or plasmid DNA (pDNA) promise high potential in gene therapy. For pharmaceutical application, however, adequate drug carriers are required. Among various concepts progressing in the market or final development, nanosized hydrogel particles may serve as novel transport media especially for siRNA. In this work, a new concept of synthesizing polymeric cationic nanohydrogels was developed, which offers a promising strategy to complex and transport siRNA into cells. For this purpose, amphiphilic reactive ester block copolymers were synthesized by RAFT polymerization of pentafluorophenyl methacrylate as reactive ester monomer together with tri(ethylene glycol)methyl ether methacrylate. In polar aprotic solvents, a self-assembly of these polymers could be observed leading to the formation of nanometer-sized polymer aggregates. The resulting superstructures were used to convert the reactive precursor block copolymers with amine-containing cross-linker molecules into covalently stabilized hydrogel particles. Detailed dynamic light scattering studies showed that the structure of the self-assembled aggregates can permanently be locked-in by this process. This method offers a new possibility to synthesize precise nanohydrogels of different size starting from various block copolymers. Moreover, via reactive ester approach, further functionalities could be attached to the nanoparticle, such as fluorescent dyes, which allowed distinct tracing of the hydrogels during complexation with siRNA or cell uptake experiments. In this respect, cellular uptake of the particles themselves as well as with its payload could be detected successfully. Looking ahead, these novel cationic nanohydrogel particles may serve as a new platform for proper siRNA delivery systems.


Subject(s)
Drug Carriers/chemistry , Hydrogels/chemistry , Nanostructures/chemistry , RNA, Small Interfering/metabolism , Amines/chemistry , Animals , Biological Transport , Cell Line , Drug Carriers/metabolism , Esters , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Conformation , Rats , Solvents/chemistry
9.
Methods Mol Biol ; 606: 439-55, 2010.
Article in English | MEDLINE | ID: mdl-20013414

ABSTRACT

By monitoring the efficiency of fluorescence resonance energy transfer of dyes attached to the different strands of siRNA, the structural integrity of the latter can be traced inside cells. Here, the experimental details of dye-labeled siRNA construction, tissue culture, and transfection with liposomally formulated siRNAs are given, as well as the conditions for confocal microscopy and an algorithm allowing the visualization of intact siRNA after image data treatment. The method allows rapid screening of different liposomal siRNA formulations, obtained by small scale dual asymmetric centrifugation with high entrapping efficiency.


Subject(s)
Fluorescence Resonance Energy Transfer/methods , Liposomes , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/analysis , Animals , Cell Line , Endothelial Cells/cytology , Fluorescence Resonance Energy Transfer/economics , Fluorescent Dyes/analysis , Humans , Liposomes/chemistry , Microinjections , Rats , Tissue Fixation , Transfection
10.
J Control Release ; 135(1): 80-8, 2009 Apr 02.
Article in English | MEDLINE | ID: mdl-19124051

ABSTRACT

Liposomal formulation of siRNA is an attractive approach for improving its delivery in vivo, shielding the RNA from nucleases and promoting tumor targeting. Here, the production of very small batch sizes of siRNA-liposomes by using the "dual asymmetric centrifugation (DAC)" technique was investigated. This new technique combines rapid and sterile liposome preparation with very high entrapping efficiencies. DAC is here presented in conjunction with a non-destructive microscale analysis based on double fluorescence labeling, which enables monitoring of siRNA integrity during the liposomal preparation. Integrity is reflected in spatial proximity of the dyes, which results in measurable fluorescence resonance energy transfer (FRET). The combination of DAC and the sensitive FRET analysis allows the handling of batch sizes down to 20 mg of conventional liposomes (CL) and sterically stabilized liposomes (SL). These were prepared in common 2 ml reaction tubes and loaded with calcein or labeled siRNA. Liposome sizes were 79+/-16 nm for CL and 109+/-9 nm for SL loaded with siRNA. Trapping efficiencies ranged from 43 to 81%, depending on batch size, enclosed compound, and liposome composition. FRET monitoring showed that the siRNA remained intact throughout DAC and that liposomal formulations protected the siRNA from nucleases. siRNA-liposomes remained stable for at least 3 months.


Subject(s)
Drug Compounding/methods , RNA, Small Interfering/administration & dosage , Centrifugation , Chromatography, High Pressure Liquid , Drug Compounding/instrumentation , Drug Stability , Fluoresceins/chemistry , Fluorescence Resonance Energy Transfer , Liposomes , Particle Size , Spectrometry, Fluorescence , Sterilization
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