Single-Molecule Imaging of an <i>in Vitro</i>-Evolved RNA Aptamer Reveals Homogeneous Ligand Binding Kinetics

Elenko, Mark P.; Szostak, Jack W.; Oijen, Antoine M. van

doi:10.1021/ja901880v

Cited by 44 publications

(50 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Single-molecule approaches to biomolecular interaction kinetics (24-26) allow for direct measurements of binding and unbinding rates (27)(28)(29). In addition, the combination of single-molecule florescence (16, 18) with single-molecule force spectroscopy (23, 30) marks itself as an especially promising tool in unbinding studies because the fluorescence readout for catalytic activity is expected to be correlated with selective, force-induced, changes of the activation barrier for unbinding (23,30).…”

mentioning

confidence: 99%

Role of substrate unbinding in Michaelis–Menten enzymatic reactions

Reuveni

Urbakh

Klafter

2014

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

245

218

View full text Add to dashboard Cite

The Michaelis-Menten equation provides a hundred-year-old prediction by which any increase in the rate of substrate unbinding will decrease the rate of enzymatic turnover. Surprisingly, this prediction was never tested experimentally nor was it scrutinized using modern theoretical tools. Here we show that unbinding may also speed up enzymatic turnover-turning a spotlight to the fact that its actual role in enzymatic catalysis remains to be determined experimentally. Analytically constructing the unbinding phase space, we identify four distinct categories of unbinding: inhibitory, excitatory, superexcitatory, and restorative. A transition in which the effect of unbinding changes from inhibitory to excitatory as substrate concentrations increase, and an overlooked tradeoff between the speed and efficiency of enzymatic reactions, are naturally unveiled as a result. The theory presented herein motivates, and allows the interpretation of, groundbreaking experiments in which existing single-molecule manipulation techniques will be adapted for the purpose of measuring enzymatic turnover under a controlled variation of unbinding rates. As we hereby show, these experiments will not only shed first light on the role of unbinding but will also allow one to determine the time distribution required for the completion of the catalytic step in isolation from the rest of the enzymatic turnover cycle.single enzyme | enzyme kinetics | renewal theory E very enzymatic reaction is composed out of two basic steps:(i) the reversible binding of a substrate molecule to the enzyme and (ii) a catalytic step which gives rise to the formation of the product (1). Controlled variation of the effective binding rate to a free enzyme is attained by altering the concentration of the substrate. The rate of product formation, also known as the turnover rate, can then be measured as a function of this concentration to show a characteristic hyperbolic dependence (2-5). The Michaelis-Menten equation is used to rationalize this observation and interpret the results (2, 6).Recent advancements in single-molecule spectroscopy have gradually made it possible to follow the stochastic activity of individual enzymes over extended periods of time (7-19). Interestingly, the hyperbolic dependence of turnover on substrate concentration remains valid even at the single-molecule level (12,14). From a theoretical perspective, this result was initially puzzling but is now considered almost universal in the sense that it can be shown to hold under a wide range of modeling assumptions (20, 21). One can thus safely say that the role of binding in Michaelis-Menten enzymatic reactions is well understood. In this paper, we consider the role of unbinding.Rapid advancements in the single-molecule technological front imply that the adaptation of existing single-molecule manipulation techniques (12-14, 18, 22, 23) to allow the measurement of enzymatic turnover under a controlled variation of unbinding rates is now within reach. Single-molecule approaches to biomolecular inter...

show abstract

mentioning

confidence: 99%

Role of substrate unbinding in Michaelis–Menten enzymatic reactions

Reuveni

Urbakh

Klafter

2014

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

245

218

View full text Add to dashboard Cite

show abstract

“…In a proof-of-principle experiment, we encapsulated biotin-coated fluorescent nanoparticles (40 nm in diameter) in oleate vesicles (containing 10 mM HPTS, in 0.2 M Na-bicine, pH 8.5), which were lysed under intense illumination in a microfluidic channel (Additional File 1 Figure S9A, B, C; Additional File 8). This process allowed the released biotin-coated fluorescent nanoparticles to attach to the streptavidin-coated surface of a microfluidic channel [26] within the illuminated region of approximately 100 μm in length (Additional File 1 Figure S9D). More generally, the ability to release vesicle-encapsulated substances in a highly spatio-temporally controlled manner provides an alternative to the chemical release of caged derivatives of small molecules [27], such as for studying bacterial chemotaxis and neuronal signaling (Additional File 1 Figure S10).…”

Section: Resultsmentioning

confidence: 99%

Exploding vesicles

Zhu

Szostak

2011

J Syst Chem

View full text Add to dashboard Cite

While studying fatty acid vesicles as model primitive cell membranes, we encountered a dramatic phenomenon in which light triggers the sudden rupture of micron-scale dye-containing vesicles, resulting in rapid release of vesicle contents. We show that such vesicle explosions are caused by an increase in internal osmotic pressure mediated by the oxidation of the internal buffer by reactive oxygen species (ROS). The ability to release vesicle contents in a rapid, spatio-temporally controlled manner suggests many potential applications, such as the targeted delivery of cancer chemotherapy drugs, and the controlled deposition of functionalized nanoparticles in microfluidic devices. Recent observations of light-triggered lysosome rupture in vivo suggest the possibility that a common mechanism may underlie light-triggered vesicle explosions and lysosome rupture. FindingsVesicles are bilayer membrane structures that encapsulate an internal aqueous compartment. Fatty acid vesicles have been studied as models of primitive cell membranes at the origin of life [1][2][3][4][5][6][7], and phospholipid vesicles (liposomes) have been widely studied as models of modern cell and organelle membranes [8,9] and as drug delivery vehicles [10][11][12]. We first observed the phenomenon of "exploding vesicles" during microscopic observations of large (approximately 4 μm in diameter) oleate vesicles containing 10 mM HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt, a water-soluble, membrane-impermeable fluorescent dye), in 0.2 M Nabicine buffer, pH 8.5. The vesicles were prepared by extrusion and dialysis, so that the fluorescent dye was present only inside the vesicles while the buffer was present in both inner and outer solutions [13]. To our surprise, we observed that these vesicles suddenly exploded shortly (approximately 0.5 sec) after being exposed to intense illumination from a metal halide lamp (estimated irradiance 2.5 W/mm 2 ), and released their encapsulated dye along with smaller internal vesicles ( Figure 1A; Additional File 1 Figure S1; Additional File 2). The actual vesicle rupture appeared to take place in < 2 ms (3 frames in a recording from a high-speed camera: Additional File 3); this estimate is an upper limit because of the time required for diffusion of the released dye away from the vesicle. We observed similar vesicle explosions using vesicles containing different internal fluorescent dyes, such as calcein and Rose Bengal (a photodynamic therapy drug).To distinguish between physical rupture and rapid permeabilization of the vesicle membrane, we labeled the vesicles with a membrane-localized dye (Rh-DHPE, Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine). Upon illumination, we observed that the vesicle membrane burst open on one side and then quickly recoiled ( Figure 1B; Additional File 4). When using slightly lower illumination intensity, we observed the gradual rupture of the multiple layers of multilamellar vesicles, starting from the outer layers and progressing to...

show abstract

“…RNA aptamer experiment details have been published. 16 The experiments described here made use of the 9-4, 10-10, and class V aptamers, 19,20 and mutants. RNA was prepared 20 with a 3Ј A 20 tail.…”

Section: Methodsmentioning

confidence: 99%

“…We illustrate the long time scale single-molecule imaging method described here with a study on RNA aptamers ͑see also Elenko et al 16 ͒. Aptamers are structured oligonucleotides that are capable of binding ligands with high specificity and affinity.…”

Section: Guanosine Triphosphate-binding Aptamers: a Case Studymentioning

confidence: 99%

Single-molecule binding experiments on long time scales

Elenko

Szostak

Oijen

2010

Review of Scientific Instruments

Self Cite

View full text Add to dashboard Cite

Single-molecule binding experiments on long time scales Elenko, Mark P.; Szostak, Jack W.; van Oijen, Antoine M. Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. We describe an approach for performing single-molecule binding experiments on time scales from hours to days, allowing for the observation of slower kinetics than have been previously investigated by single-molecule techniques. Total internal reflection fluorescence microscopy is used to image the binding of labeled ligand to molecules specifically coupled to the surface of an optically transparent flow cell. Long-duration experiments are enabled by ensuring sufficient positional, chemical, thermal, and image stability. Principal components of this experimental stability include illumination timing, solution replacement, and chemical treatment of solution to reduce photodamage and photobleaching; and autofocusing to correct for spatial drift. © 2010 American Institute of Physics. ͓doi:10.1063/1.3473936͔ I. SINGLE-MOLECULE MICROSCOPY FOR BINDINGBinding is arguably the most fundamental of biomolecular interactions, and as such has been investigated using a wide array of methods. Canonical solution-phase methods are often limited to nonequilibrium measurements, are restricted in the rate and K d ranges they can access, and provide only ensemble-averaged molecular information. As such, it can be challenging to capture the fine structure of molecular behavior with bulk-phase methods. We seek to take advantage of the blooming of single-molecule techniques that have already enabled the exploration of previously inaccessible biomolecular phenomena, including enzymatic memory, molecular motor motion, folding, conformational change, and catalysis. [1][2][3][4] The study of these phenomena at the ultimate level of sensitivity, that of a single molecule, allows for the observation of variations between and within single molecules under equilibrium conditions.Single-molecule experiments have typically explored molecular interactions taking place at short time scales up to seconds or a few minutes. These typical time scales place a severe limit on the kinetic space that can be explored. For a diffusion-limited ͑10 8 M −1 s −1 ͒ bimolecular interaction, a very tight K d of 10 nM translates to an off rate of 1 s −1 , resulting in a duration of the binding cycle that is readily captured in a short time frame. Many bimolecular interactions of interest, however, display nondiffusion limi...

show abstract

Single-Molecule Imaging of an in Vitro-Evolved RNA Aptamer Reveals Homogeneous Ligand Binding Kinetics

Cited by 44 publications

References 14 publications

Role of substrate unbinding in Michaelis–Menten enzymatic reactions

Role of substrate unbinding in Michaelis–Menten enzymatic reactions

Exploding vesicles

Single-molecule binding experiments on long time scales

Contact Info

Product

Resources

About