Determination of the turnover number of the restriction endonuclease EcoRI using evanescent wave technology

Bier, Frank F.; Kleinjung, Frank; Schmidt, Peter M.; Scheller, Frieder W.

doi:10.1007/s00216-001-1216-4

Cited by 10 publications

(3 citation statements)

References 9 publications

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“…Many of these are fluorescence-based and include fluorescence resonance energy transfer (FRET) 1 (4), dual-color fluorescence cross-correlation spectroscopy (5,6), fluorescence anisotropy combined with FRET (7), or the use of a fluorophore and a quencher at the ends of a nucleic acid hairpin (8). Alternative approaches include the use of immobilized nucleic acids on evanescent wave sensors (9,10) and ferrocene-modified oligonucleotides coupled with electrochemical detection (11). All of these approaches require modification of the substrate with either a label or a means of immobilization on a surface.…”

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confidence: 99%

Restriction Enzyme Kinetics Monitored by UV Linear Dichroism

et al. 2006

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The use of linear dichroism (LD) spectroscopy for biological applications has been brought to the forefront recently by our development of thermostated microvolume Couette cells. We present a method for following the digestion of DNA by restriction endonucleases in real time without the use of any extrinsic dyes or labels. This is accomplished using linear dichroism spectroscopy (the differential absorbance of light polarized parallel and perpendicular to the sample orientation axis). The differential absorbance signal depends on the degree of alignment of the molecules. In this case the DNA is aligned by Couette flow (flowing the solution in the annular gap between two concentric cylinders), and we monitor the increase in alignment upon linearization of a circular DNA molecule. In addition, we observe a decrease in alignment upon further digestion and subsequent shortening of the DNA. Ten enzymes were investigated: seven enzymes with a single cut site (EcoRI, KpnI, NdeI, NotI, NruI, SmaI, XbaI), two enzymes with two cut sites (BstZ17I, EagI), and one enzyme with no cut site (ClaI). LD, as implemented in this new assay, is broadly applicable across a wide range of DNA-modifying enzymes and compounds and, as such, is a useful addition to the toolbox of biological characterization.

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Restriction Enzyme Kinetics Monitored by UV Linear Dichroism

et al. 2006

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“…The endonucleases, because of their capability to bind and cleave specific DNA sequences, also play a key role in DNA recombinant and cloning methods (1,2). The EcoR1-DNA equilibria and reaction dynamics have been probed extensively by a number of established methods, such as, but not limited to: FRET (3)(4)(5), gel electrophoresis (6)(7)(8)(9)(10)(11)(12)(13)(14)(15), surface plasmon resonance (16), microfluidic trapping (17), two-color cross-correlation spectroscopy (18), and isothermal calorimetry (19).…”

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Binding and cleavage of DNA with the restriction enzyme EcoR1 using time-resolved second harmonic generation

Doughty

Kazer

Eisenthal

2011

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

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The binding of EcoR1 to a 90-bp DNA duplex attached to colloidal microparticles and the subsequent cleavage by the enzyme was observed in real time and label-free with time-resolved second harmonic (SH) spectroscopy. This method provides a unique way to investigate biomolecular interactions based on its sensitivity to changes in structure and electrical charge on formation of a complex and subsequent dynamics. The binding of EcoR1 to the recognition sequence in DNA appears as a rapid increase in the SH signal, which is attributed to the enzyme-induced change in the DNA conformation, going from a rod-like to a bent shape. In the presence of the cofactor Mg 2 , the subsequent decay in the SH signal was monitored in real time as the following processes occurred: cleavage of DNA, dissociation of the enzyme from the DNA, and diffusion of the 74-bp fragment into the bulk solution leaving the 16-bp fragment attached to the microparticle. The observed decay was dependent on the concentration of Mg 2 , which functions as a cofactor and as an electrolyte. With SH spectroscopy the rehybridization dynamics between the rehybridized microparticle bound and free cleaved DNA fragments was observed in real time and label-free following the cleavage of DNA. Collectively, the experiments reported here establish SH spectroscopy as a powerful method to investigate equilibrium and time-dependent biological processes in a noninvasive and label-free way.cleavage kinetics | DNA-endonuclease | rehybridization kinetics | nonspecific binding T he highly selective binding of proteins to specific nucleotide sequences in DNA is an essential step in many biological processes, including gene regulation, gene expression, and DNA hybridization (1). The family of type II restriction enzymes, which includes EcoR1, have been, and continue to be, of major importance in serving as model systems of protein-DNA interactions. The endonucleases, because of their capability to bind and cleave specific DNA sequences, also play a key role in DNA recombinant and cloning methods (1, 2). The EcoR1-DNA equilibria and reaction dynamics have been probed extensively by a number of established methods, such as, but not limited to: FRET (3-5), gel electrophoresis (6-15), surface plasmon resonance (16), microfluidic trapping (17), two-color cross-correlation spectroscopy (18), and isothermal calorimetry (19).The essential characteristic of the method used here is the application of second harmonic (SH) spectroscopy to selectively probe the interface of a polystyrene carboxylate (PSC) microparticle, to which target DNA molecules have been attached. Significant effort and progress has been made in hybridizing artificial colloidal nano-and micromaterials with naturally occurring biological reagents to build self-assembled superstructured materials (20-26), and to develop biologically coded probes for protein detection and disease treatment (27,28). The present work builds on these pioneering studies, and on studies of DNA supported on flat surfaces using SH and sum-frequ...

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“…Enzymatic reactions on immobilised substrates or templates might also be observed by use of a flowthrough scanning device. As has been shown recently, single measurements with optical biosensors for a single type of substrate [9], parallel detection, and comparison of a variety of substrates or templates are now accessible in a single experiment. To demonstrate the power of the approach, we chose a restriction endonuclease.…”

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Real-time analysis on microarrays

Bier

Ehrentreich‐Förster

Schmidt

2004

Analytical and Bioanalytical Chemistry

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To obtain the maximum amount of information from the smallest amount of a sample is one of the major goals of analytical science. In molecular biology and all molecularbased life sciences there is also a broad demand for highly parallel analysis. Microarray technology is the answer to this demand. It enables massive parallel determination and multiple measurement of a variety of binding events to be performed simultaneously. It has, in addition, the advantage of requiring modest investment of labour and might, therefore, save much time and be automated easily. Microarrays usually consist of many microscopic spots each containing identical molecules, i.e. receptors, probes, or targets. The number of spots can vary from less than one hundred to several hundred thousand. The molecules are attached to a solid support which can be made from glass or a polymer. The primary task of a microarray experiment is to detect many binding events simultaneously.Most applications use fluorescence as a label to detect the binding events. Before the experiment the sample must be labelled by means of a suitable fluorochrome. Binding is achieved in a separate incubation step and the final result is acquired after drying of the microarray. Modern microarray readers usually, therefore, acquire information about the fluorescence intensity at a given time of the binding process. Many applications have been reported during the past two years, mainly in the area of transcription analysis [1,2, 3]. Most experiments compare two states of a cell type, under different conditions, to identify the relevant activated genes. Progress into more analytical and diagnostic applications is still slow, because quantitation of the results still is a problem and fabrication methods are not yet sufficiently reliable to enable production of larger series. The fluorescence intensity measured in microarray experiments represents the amount of bound, i.e. labelled, analyte; this depends on the concentration in the applied solution and on the affinity of the binding partners and the time allowed for binding. It is not usually possible to differentiate among these influencing factors in the usual arrangement. The physical situation of the microarray experiment can, moreover, withstand high selectivity when the association rate constant dominates the result, as has been outlined recently [4]. From biosensor technology we have learned to analyse complete binding processes on surface-immobilised receptors by combining optical or electronic signal transduction with microfluidics. To overcome the limitations of microarray technology developments are under way to facilitate measurement of binding kinetics in the microarray format. Homogeneous sample flow over the whole microarray is one technological problem that has recently been solved in our laboratory by use of computer-aided simulations. Finite element calculations were used to optimise the design of the flow cells [5].The scientific community is still waiting for the successful expansion of label-free means of d...

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Determination of the turnover number of the restriction endonuclease EcoRI using evanescent wave technology

Cited by 10 publications

References 9 publications

Restriction Enzyme Kinetics Monitored by UV Linear Dichroism

Restriction Enzyme Kinetics Monitored by UV Linear Dichroism

Binding and cleavage of DNA with the restriction enzyme EcoR1 using time-resolved second harmonic generation

Real-time analysis on microarrays

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