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Nanopory : budowa, właściwości, modele, zastosowania

Stachiewicz A., Molski A.

Wiadomości Chemiczne

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2013

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[Z] 67, 3-4

277--302

EN

Nanopores are small (1–100 nm diameter) holes/channels formed in biological membranes (Fig. 1) or fabricated in synthetic materials (Fig. 2). Permeation of ions and small molecules through nanopores is common in biological systems. The first experiments where nanopores were used as single-molecule sensors were performed in the 90s [1, 2]. The detection principle is based on a monitoring of an ionic current passing through a nanopore as an electric field is applied across the membrane. Electrically charged particles (e.g. DNA ) move in the electric field and block the ionic current as they pass through the nanopore. A sudden drop of the ionic current signals a single-molecule translocation event (Fig. 3–5). Nanopore sensors can give an information about the analyte: its size, structure and bonds stability. Today, a major topic of interest is the possibility of nanopore DNA sequencing. In this work we present an introduction to nanopore technology and to current research related to potential nanopore applications. First, we describe biological and synthetic nanopores: their structure and methods of fabrication. Next, different modes of nanopore experiments are presented. In the third section, we focus on theoretical models and simulations of nanopores. Finally, we present future perspectives for applications with particular reference to DNA sequencing.

2

Enzymologia pojedynczych cząsteczek RNA : wykorzystanie techniki FRET w badaniach zwijania się i dynamiki konformacyjnej rybozymów

Hajdziona M., Molski A.

Wiadomości Chemiczne

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2010

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[Z] 64, 3-4

173-193

EN

Ribozymes are biologically important macromolecules that play a crucial role in cell metabolism and functions. Knowledge of folding and catalytic properties of ribozymes can be useful in biotechnology and medicine. In this work, we present a review of single-molecule RNA enzymology with particular emphasis on folding and catalysis observed with single-molecule FRET. Single-molecule spectroscopy provides insight into behaviors of individual molecules without averaging inherent in bulk measurements. In the first section we introduce ribozymes as RNA enzymes [1, 2]. In the second section, we present the structure of RNA molecules and examples of reaction mechanisms (Fig. 1) and function of different types of ribozymes (Fig. 2–5). Next, we review single-molecule FRET spectroscopy (Fig. 6, 7, Eqs. 1, 2). In the fourth section, we present examples of folding dynamics of ribozymes. In the fifth part, we focus on ribozyme catalysis (Fig. 8, 9). We discuss the coupling of conformational dynamics with catalytic reactions. In the last part we present methods of data analysis that can be used to obtain the kinetic rates from single molecule FRET experiments (Fig. 10).

3

90 lat chemii na Uniwersytecie w Poznaniu

Molski A., Nowak I.

Analityka : nauka i praktyka

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2009

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nr 2

32-34

4

Aktywność katalityczna pojedynczych cząsteczek enzymów

Jakubaszek J., Molski A.

Wiadomości Chemiczne

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2008

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[Z] 62, 7-8

635-658

EN

As early as in 1961, it has been demonstrated that turnovers of individual enzyme molecules can be detected [1]. In the nineties, advances in single-molecule methods, in particular in confocal microscopy (Fig. 1), made it possible to monitor more closely enzymatic turnovers at a single-molecule level [2-5]. This led to the discoveries of static disorder and dynamic disorder, and closely related memory effects in enzymatic turnovers [6-8]. Differences in activity of individual molecules of the same enzyme are called static disorder. Time-dependent fluctuations of enzymatic activity are called dynamic disorder. One manifestation of dynamic disorder is the fact that consecutive enzymatic turnovers are not statistically independent, which is called "memory effect". It is believed that static dis-order and dynamic disorder are related to conformational dynamics of enzyme molecules. In this review we discuss current issues of single-molecule enzymology, in particular kinetic effects that are specific to single-enzyme measurements. First we review the conceptual basis of single-enzyme kinetics and the initial work on single enzymes. We focus on the ping-pong mechanism of bisubstrate enzyme reactions (Eqs. (1) and (2)), and consider fluorescence trajectories (Figs. 2 and 3) associated with enzymatic turnovers. Two cases are distinguished. In the first, fluorescence comes from an enzyme molecule and fluorescence intensity jumps called blinking carry information on enzymatic activity. Jumps between a fluorescent (on) and non-fluorescent (off) states (Eqs. (4), (5), and Fig. 2) indicate the moments when the photophysical state of an enzyme changes during enzymatic turnovers. In the second case, fluorescence comes from product molecules whereas enzyme and substrate are non-fluorescent. Fluorescence bursts on a trajectory indicate the moments when non-fluorescent substrate molecules are converted into fluorescent product molecules that subsequently diffuse away from the detection volume (Eqs. (8), (9) and Fig. 3). In Section 1 we present selected experiments implying the effect of conformational dynamics on enzymatic kinetics. In Section 1.1, we discuss cholesterol oxydase and dihydroorotate dihydrogenase as examples of enzymes whose on-off fluorescence blinking is caused by chemical reactions at the enzyme active site. In Section 1.2 we discuss ?-lactosidase and lipase B as enzymes which turnovers can generate fluorescent products from suitably chosen non-fluorescent substrates. In Section 2, we review modeling and simulations of single-enzyme data. The aim of data modeling is to gain insight into single-enzyme activity through analysis of models of increasing complexity. Phenomenological models attempt to capture the essence of single-enzyme kinetics without going into molecular details. If a model is simple enough it may allow analytical solutions. For instance, a simple model of on-off blinking is given in Scheme (25). This scheme is capable of reproducing memory effect that can be visualized by a two-dimensional histogram of consecutive on and off times (Figs. 5a and 5b). Finally, in the last section we present an outlook for single-molecule enzymology.

5

Spektroskopia pojedynczych fluoroforów

Molski A.

Wiadomości Chemiczne

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2006

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[Z] 60, 7-8

449-465

EN

This article is an introduction to single-fluorophore spectroscopy (SFS). The basic phenomenon seen in SFS is the fluctuations of fluorescence intensity of single nano-objects (dyes, biopolymers, nano-crystals, aggregates). These fluctuations are called fluorescence blinking and carry information on the dynamics of a fluorophore and its surroundings. In the simplest case the fluorescence trajectory jumps between two levels, a bright one (on) and a dark one (off). In this article on-off fluorescence blinking associated with triplet states of organic dyes, excitation of quantum dots, and enzymatic reactions at room temperatures are reviewed. The cited literature indicates current problems and may serve as an introduction to modelling and simulations of SFS.