Quantitative Mapping of Endosomal DNA Processing by Single Molecule Counting

Prakash, Ved; Tsekouras, Konstantinos; Venkatachalapathy, Muthukumaran; Heinicke, Laurie A.; Pressé, Steve; Walter, Nils G.; Krishnan, Yamuna

doi:10.1002/anie.201811746

Cited by 18 publications

(16 citation statements)

References 27 publications

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“…CellMask™ reagents and TMR-Dextran were purchased from molecular probes/Life Technologies (USA). Maleylated BSA (mBSA) and fluorescent transferrin (Tf-Alexa546) were conjugated according to previously published protocols 18 , 37 , 51 . Pharmacological Lysosomal modulators were purchased from Cayman Chemical (USA).…”

Section: Methodsmentioning

confidence: 99%

A DNA-based voltmeter for organelles

et al. 2020

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The role of membrane potential in most intracellular organelles remains unexplored because of the lack of suitable tools. Here, we describe a fluorescent DNA-nanodevice that reports absolute membrane potential and can be targeted to organelles in live cells. This nanodevice, denoted Voltair , bears a voltage-sensitive fluorophore, a reference fluorophore for ratiometry, and acts as an endocytic tracer. Using Voltair we could measure the membrane potential of different organelles in situ in live cells. Voltair can potentially guide the rational design of biocompatible electronics as well as expand our understanding of how membrane potential regulates organelle biology.

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Section: Methodsmentioning

confidence: 99%

A DNA-based voltmeter for organelles

et al. 2020

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“…Thus, our results suggest that the DNA/DNA duplex is delivered to the liver as a double-stranded structure and then gets degraded more rapidly than the DNA/RNA heteroduplex. One possible explanation is that the DNA/DNA duplex and DNA/RNA heteroduplex are digested in different intracellular compartments by DNases 20 , 35 , 36 and RNases, 37 respectively. Moreover, despite the rapid degradation of Toc-HDO (coDNA), the initiation timing of RNA suppression did not differ from that of Toc-HDO (coRNA).…”

Section: Discussionmentioning

confidence: 99%

Efficient Gene Suppression by DNA/DNA Double-Stranded Oligonucleotide In Vivo

et al. 2021

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We recently reported the antisense properties of a DNA/RNA heteroduplex oligonucleotide consisting of a phosphorothioate DNA-gapmer antisense oligonucleotide (ASO) strand and its complementary phosphodiester RNA/phosphorothioate 2 0 -Omethyl RNA strand. When a-tocopherol was conjugated with the complementary strand, the heteroduplex oligonucleotide silenced the target RNA more efficiently in vivo than did the parent single-stranded ASO. In this study, we designed a new type of the heteroduplex oligonucleotide, in which the RNA portion of the complementary strand was replaced with phosphodiester DNA, yielding an ASO/DNA double-stranded structure. The ASO/DNA heteroduplex oligonucleotide showed similar activity and liver accumulation as did the original ASO/RNA design. Structure-activity relationship studies of the complementary DNA showed that optimal increases in the potency and the accumulation were seen when the flanks of the phosphodiester DNA complement were protected using 2 0 -O-methyl RNA and phosphorothioate modifications. Furthermore, evaluation of the degradation kinetics of the complementary strands revealed that the DNA-complementary strand as well as the RNA strand were completely cleaved in vivo. Our results expand the repertoire of chemical modifications that can be used with the heteroduplex oligonucleotide technology, providing greater design flexibility for future therapeutic applications.

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“…Learning the number of fluorophores within an ROI is a key step toward unraveling processes below the diffraction limit [5,12,2,23,24,25,26,27,28,29,30,31,32,33,34,10,11,12,13,14,15,16,17,18,19,20,21,56]. In order to go beyond the state of the art and enumerate as many as five times more fluorophores, we must accurately account for all the following: 1) fluorophore photophysics; 2) models for shot and detector noise; 3) provide full credible intervals (error bars) about the estimates; and 4) exploit every data point available without pre-or post-processing.…”

Section: Discussionmentioning

confidence: 99%

“…Upon illumination, most fluorophores will start in their bright state, giving rise to an initially high brightness. Subsequently, one-byone, fluorophores will blink and photobleach, causing step-like transitions between brightness levels until all fluorophores are photobleached [5,12,2,23,24,25,26,27,28,29,30,31,32,33,34]. A cartoon depicting this process is laid out in figure 2.…”

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Diffraction-Limited Molecular Cluster Quantification with Bayesian Nonparametrics

Sgouralis

Bryan

Pressé

2020

Preprint

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Determining the number of biomolecules within a small, diffraction-limited spot, is key to understanding intermolecular interactions that form the basis of all of information processing relevant to life. To enumerate the number of biomolecules within such a small region of interest (ROI), it is possible to illuminate an ROI containing a collection of fluorescently labeled biomolecules and observe the ROI's brightness over time. As most fluorescent labels (fluorophores) are initially in a fluorescently active state, the brightness diminishes in a step-like pattern as fluorophores photobleach one at a time. Naively, by counting steps, one could determine the number of fluorophores present. However, this analysis is complicated by photon shot noise, camera noise that amplifies intrinsic photon shot noise and fluorophore photophysics (i.e., states with variable emission visited by fluorophores). Current state of the art can typically enumerate as many as 20 fluorophores reliably. Yet in many biophysics problems, fluorophore numbers can vastly exceed 20 within an ROI. For this reason, we develop a method to learn the number of fluorophores alongside other parameters required to achieve high counts simultaneously and self-consistently (e.g., fluorophore photophysical rate parameters and camera parameters). As the number of fluorophores is initially unknown and may be very large, and as not all fluorophores may initially be emitting photons, we cannot rely on a traditional (parametric) Bayesian framework. As such, to achieve our goal of exceeding the state of the art by a factor of about 5, we must abandon the parametric Bayesian paradigm and invoke novel tools within Bayesian nonparametrics never previously used in step counting by photobleaching.

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Quantitative Mapping of Endosomal DNA Processing by Single Molecule Counting

Abstract: This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record.

Cited by 18 publications

References 27 publications

A DNA-based voltmeter for organelles

A DNA-based voltmeter for organelles

Efficient Gene Suppression by DNA/DNA Double-Stranded Oligonucleotide In Vivo

Diffraction-Limited Molecular Cluster Quantification with Bayesian Nonparametrics

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