Current Pharmaceutical Biotechnology - Volume 5, Issue 3, 2004

The collection of articles published under the motto “The way down from single genes and proteins to single molecules” is covering the results of groundbreaking developments of the recent years. They include single molecule detection yielding ultimate sensitivity and closely linked to the analysis of molecular noise by correlation spectroscopy. Several important examples from the field of nucleic acid as well as protein analysis as diagnostic tools are presented as well as the impact on the studies of single cells and biomembranes. The development of high sensitivity analysis at the single molecule level has in turn prompted the development of high-throughput protocols nowadays used in drug screening. In the attempt to make the vast and still growing knowledge of the human genome available for understanding the ultimate cause of disease and their therapy, these four special issues will fulfill a very important goal.

The present review gives a short summary on techniques useful for single molecule research, describes experiments on in vitro single molecule detection and reactions of single molecules and finally reports on the behavior of single molecules and single virus particles in living cells. One experiment on single molecule enzyme kinetics of lactate dehydrogenase, an enzyme used in the diagnosis of heart attacks and one experiment on restriction analysis of individual DNa molecules are described in some detail. Where it is possible, the relevance to pharmacology and biomedicine is emphasized, often as a perspective or suggestion for experiments, since in this young field of science a not too large variety of experiments have indeed already been devoted directly to drug action.

Protein conformational dynamics, often associated with static and dynamic inhomogeneities, plays a crucial role in biomolecular functions. It is extremely difficult to characterize such inhomogeneous dynamics in an ensembleaveraged measurement, especially when the protein involves in a multiple-step complex chemical reaction, such as an enzymatic reaction. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics in real time under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms. In this minireview, we discuss our recent studies on single-molecule enzymatic reaction dynamics and protein conformational dynamics of the T4 lysozyme hydrolyzation reaction of a polysaccharide by a combined approach of single-molecule spectroscopy, molecular dynamics simulation, and theoretical modeling. Detailed characterization of the complex enzymatic reaction dynamics identified the nature of the inhomogeneity and revealed multiple intermediate conformational states associated with the enzyme-substrate complex formation in the multiple-step enzymatic reaction.

Recently developed single-molecule spectroscopy (SMS) permits the analysis of fluorescent mixtures one molecule at a time. SMS methods provide the means to make rapid measurements on small, complex samples without the need for separations and target amplification enabling a new class of ultrasensitive nucleic acid assays. Here we give a brief overview of the current state of the art of SMS nucleic acid analysis and discuss ongoing work in our laboratory on two-color single-molecule fluorescence detection of specific nucleic acid sequences. In the future, two-color SMS nucleic acid assays will be used for a variety of applications including: gene expression analysis, disease detection and genomics.

We have devised a new technique based on single fluorescent molecule detection for the analysis of specific sequences of DNA. The method consists of synthesizing a fluorescent reporter molecule using a polymerase extension reaction and labeled nucleotides. The fluorescent reporter products are analyzed in a laser-based single-molecule detection system. We have applied this method to the detection of pUC19 and Bacillus anthracis DNA targets. We expect that this method will have applications in rapid detection and identification of DNA from pathogens as well as other sources, and that it will be used for processing of large number of samples in a short period of time.

This mini-review describes how single-molecule sensitive fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET) reactions can be successfully applied to monitor conformational dynamics in biopolymers. Single-pair FRET experiments are ideally suited to study conformational dynamics occurring on the nanometer scale, e.g. during protein folding or unfolding. In contrast, conformational dynamics with functional significance, for example occurring in enzymes at work, often appear on much smaller spatial scales of up to several Angströms. Our results demonstrate that selective PET-reactions between fluorophores and amino acids or DNA nucleotides represent a versatile tool to measure small-scale conformational dynamics in biopolymers on a wide range of time scales, extending from nanoseconds to seconds, at the single-molecule level. That is, the monitoring of conformational dynamics of biopolymers with temporal resolutions comparable to those within reach using new techniques of molecular dynamic simulations. Furthermore, we demonstrate that the strong distance dependence of charge separation reactions on the sub-nanometer scale can be used to develop conformationally flexible PET-biosensors. These sensors enable the detection of specific target molecules in the sub-picomolar range and allow one to follow their molecular binding dynamics with temporal resolution.

We present novel technical features and results from a two channel confocal fluorescence lifetime microscope, which allows to efficiently investigate fluorescence dynamics down to the single molecule level. The MicroTime 200 time-resolved fluorescence microscope offers a multicolor excitation where different picosecond diode lasers are used. For imaging and positioning purposes we utilize a compact Piezo scanner which allows, due to a novel scanning algorithm and synchronisation technique, a superior movement and positioning accuracy. The data acquisition is completely based on time-correleted single photon counting, where every photon is detected and stored individually with its specific timing information (Time-Tagged Time-Resolved mode). This multiparameter data acquisition scheme offers the opportunity to analyse the parameter dependencies in a multitude of different ways. Standard intensity analysis can be used to reconstruct 2D-images or the temporal evolution (time trace) of the fluorescence of a single spot. The information from the two distinct detector channels additionally allows to investigate the polarisation of the emitted light or its spectral composition, for example for analysis of Fluorescence Resonance Energy Transfer (FRET). The timing information down to a picosecond scale offers the possibility not only to reconstruct fluorescence decay constants of each pixel for the purpose of Fluorescence Lifetime Imaging (FLIM) but also to analyze the fluorescence fluctuation correlation function of any single spot of interest. The flexible multichannel detector scheme enables in this case also a cross-correlation between spectrally separated parts of the emission light, or even identical parts of the fluorescence to eliminate detector artifacts. The photon arrival coincidence analysis can also be expanded in the sub-ns range to study fluorescence antibunching in the fluorescence emission of single molecules. The abilty of combining these different pieces of temporal information allows the construction of extremely powerful analysis methods and assays. We demonstrate a variety of these capabilities with results obtained from fluorescently labeled latex beads, biological samples, and single molecules excited in the blue or red wavelength region.

Recent advances in the development of new microscopical techniques with single-molecule sensitivity have given access to essentially new types of information on biological systems. In this review, basic methodological concepts of ultra-sensitive microscopy are presented and characterized, with focus on their applicability for a bioanalytical instrument. Measurements on artificial lipid bilayers were used to evaluate the feasibility of this novel technology. First examples of single molecule microscopy on cell membranes revealed new basic insights into the lateral organization of the plasma membrane.