Hybridization kinetics is different inside cells

Schoen, Ingmar; Krammer, Hubert; Braun, Dieter

doi:10.1073/pnas.0901313106

Cited by 92 publications

(119 citation statements)

References 45 publications

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“…Interestingly, all the fluorescence decay curves overlapped ( Figure 2B) with a decay rate of 0.63 min -1 , consistent with the report by Schoen et al on molecular beacon hybridization in PEG. 48 Based on this observation, we reason that the macromolecular crowding effect might not be important on DNA hybridization kinetics since the excluded volume change should be similar in the presence and absence of AuNPs. Instead, we propose that PEG might block non-specific DNA/AuNP interactions and thus accelerate DNA hybridization.…”

Section: Resultsmentioning

confidence: 99%

Effects of Polyethylene Glycol on DNA Adsorption and Hybridization on Gold Nanoparticles and Graphene Oxide

et al. 2012

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Understanding the interface between DNA and nanomaterials is crucial for rational design and optimization of biosensors and drug delivery systems. For detection and delivery into cells, where high concentrations of cellular proteins are present, another layer of complexity is added. In this context, we employ polyethylene glycol (PEG) as a model polymer to mimic the excluded volume effect of cellular proteins and test its effect on DNA adsorption and hybridization on gold nanoparticles (AuNPs) and graphene oxide (GO), both of which show great promise for designing intracellular biosensors and drug delivery systems. We show that PEG 20,000 (e.g. 4%) accelerates DNA hybridization to DNAfunctionalized AuNPs by 50-100%, but this enhanced hybridization kinetics has not been observed with free DNA. Therefore, this rate enhancement is attributed to the surface blocking effect by PEG instead of the macromolecular crowding effect. On the other hand, DNA adsorption on citrate-capped AuNP surfaces is impeded even in the presence of a trace level (i.e., parts-per-billion) of PEG, confirming PEG competes with DNA for surface binding sites. Additional insights have been obtained by studying the adsorption of a thiolated DNA and a peptide nucleic acid. In these cases, the steric effects of PEG to impede adsorption are observed. Similar observations have also been made with GO. Therefore, PEG may be used as an effective blocking agent for both hydrophilic AuNP and for GO that also contains hydrophobic domains.

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Section: Resultsmentioning

confidence: 99%

Effects of Polyethylene Glycol on DNA Adsorption and Hybridization on Gold Nanoparticles and Graphene Oxide

et al. 2012

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“…Measurements of bimolecular reaction kinetics for hybridization of short DNA strands [84] and protein-protein association [85] in living HeLa cells show that intracellular crowding changes reaction rates up to several-fold as compared with reactions in buffer. Here we discuss quantitatively these rates.…”

Section: Association Kinetics In Living Cellsmentioning

confidence: 99%

“…Schoen et al [84] studied kinetics of hybridization of short DNA chains at two different lengths 12-and 16-bp. The sequences of DNA were chosen to minimize specific interactions with other nucleic acids in cells and dsDNAs were delivered into cells by lipofection.…”

Section: Association Kinetics In Living Cellsmentioning

confidence: 99%

“…The kinetics of opening/closing of DNA due to temperature oscillations driven by laser were measured by Förster resonance energy transfer (FRET) method. Non-monotonic change of association kinetics was determined in vivo, i.e., acceleration of association of the 16-mers when compared to in vitro data (2.9 × In vitro data point to non-diffusion limited hybridization of these DNA fragments studied by Schoen et al [84]. Moreover, the kinetics of associations is slightly enhanced in vitro with crowding agents such as Ficoll-70 and dextrans [84] pointing that these agents cause depletion interactions and speed-up the binding.…”

Section: Association Kinetics In Living Cellsmentioning

confidence: 99%

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The effect of macromolecular crowding on mobility of biomolecules, association kinetics, and gene expression in living cells

et al. 2014

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We discuss a quantitative influence of macromolecular crowding on biological processes: motion, bimolecular reactions, and gene expression in prokaryotic and eukaryotic cells. We present scaling laws relating diffusion coefficient of an object moving in a cytoplasm of cells to a size of this object and degree of crowding. Such description leads to the notion of the length scale dependent viscosity characteristic for all living cells. We present an application of the length-scale dependent viscosity model to the description of motion in the cytoplasm of both eukaryotic and prokaryotic living cells. We compare the model with all recent data on diffusion of nanoscopic objects in HeLa, and E. coli cells. Additionally a description of the mobility of molecules in cell nucleus is presented. Finally we discuss the influence of crowding on the bimolecular association rates and gene expression in living cells.

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“…This may alter the mobility, folding, stability, and association rates of several proteins inside the cell. Molecular crowding has received little attention in living cells, although it is usually invoked to explain why biochemical reactions rates may vary in vivo and in vitro (26)(27)(28)(29). For instance, the diffusion coefficient of the green fluorescent protein is typically 10 times lower in cells than in vitro (30).…”

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confidence: 99%

Severe osmotic compression triggers a slowdown of intracellular signaling, which can be explained by molecular crowding

Miermont

Waharte

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

183

169

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Regulation of the cellular volume is fundamental for cell survival and function. Deviations from equilibrium trigger dedicated signaling and transcriptional responses that mediate water homeostasis and volume recovery. Cells are densely packed with proteins, and molecular crowding may play an important role in cellular processes. Indeed, increasing molecular crowding has been shown to modify the kinetics of biochemical reactions in vitro; however, the effects of molecular crowding in living cells are mostly unexplored. Here, we report that, in yeast, a sudden reduction in cellular volume, induced by severe osmotic stress, slows down the dynamics of several signaling cascades, including the stress-response pathways required for osmotic adaptation. We show that increasing osmotic compression decreases protein mobility and can eventually lead to a dramatic stalling of several unrelated signaling and cellular processes. The rate of these cellular processes decreased exponentially with protein density when approaching stalling osmotic compression. This suggests that, under compression, the cytoplasm behaves as a soft colloid undergoing a glass transition. Our results shed light on the physical mechanisms that force cells to cope with volume fluctuations to maintain an optimal protein density compatible with cellular functions.

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Hybridization kinetics is different inside cells

Abstract: It is generally expected that the kinetics of reactions inside livingDNA ͉ in vivo ͉ molecular crowding ͉ temperature oscillation ͉ optical lock-in microscopy

Cited by 92 publications

References 45 publications

Effects of Polyethylene Glycol on DNA Adsorption and Hybridization on Gold Nanoparticles and Graphene Oxide

Effects of Polyethylene Glycol on DNA Adsorption and Hybridization on Gold Nanoparticles and Graphene Oxide

The effect of macromolecular crowding on mobility of biomolecules, association kinetics, and gene expression in living cells

Severe osmotic compression triggers a slowdown of intracellular signaling, which can be explained by molecular crowding

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