Current Pharmaceutical Biotechnology, Vol. 5, No. 3, 2004
Contents
The Way Down from Single Genes and Proteins to Single Molecules
Single
Molecule Techniques for Biomedicine and Pharmacology Pp.243-259
K.O.
Greulich
Single-Molecule
Spectroscopy Studies of Conformational Change Dynamics in Enzymatic Reactions Pp. 261-269
H.P.
Lu
Single-Molecule
Spectroscopy for Nucleic Acid Analysis: A New Approach for Disease Detection
and Genomic Analysis Pp. 271-278
P.M.
Goodwin, Rhiannon L. Nolan and Hong Cai
Applications
of Single-Molecule Detection to the Analysis of Pathogenic DNA Pp. 279-284
Oana
Marina and Alonso Castro
Using
Photoinduced Charge Transfer Reactions to Study Conformational Dynamics of
Biopolymers at the Single-Molecule Level Pp. 285-298
H.
Neuweiler and M. Sauer
Time-Resolved
Confocal Fluorescence Imaging and Spectrocopy System with Single Molecule
Sensitivity and Sub-Micrometer Resolution Pp. 299-308
M.
Wahl, F. Koberling, M. Patting, H. Rahn and R. Erdmann
Ultra-Sensitive
Fluorescence Reader for Bioanalysis
Pp. 309-319
Jan
Hesse, Max Sonnleitner and Gerhard J. Schutz
Abstracts
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to top]
Single Molecule Techniques for
Biomedicine and Pharmacology
K.O. Greulich
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.
[Back
to top]
Single-Molecule Spectroscopy Studies of
Conformational Change Dynamics in Enzymatic Reactions
H.P.
Lu
[Back
to top]
Single-Molecule Spectroscopy for
Nucleic Acid Analysis: A New Approach for Disease Detection and Genomic
Analysis
P.M. Goodwin, Rhiannon L. Nolan and Hong Cai
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.
[Back to
top] Applications of Single-Molecule Detection to the Analysis of Pathogenic
DNA
Oana
Marina and Alonso Castro
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.
[Back to
top] Using Photoinduced Charge Transfer Reactions to Study
Conformational Dynamics of Biopolymers at the Single-Molecule Level
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.
[Back to
top] Time-Resolved Confocal Fluorescence Imaging and Spectrocopy System
with Single Molecule Sensitivity and Sub-Micrometer Resolution
M. Wahl, F. Koberling, M. Patting, H. Rahn and R. Erdmann
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.
[Back to
top] Ultra-Sensitive Fluorescence Reader for Bioanalysis
Jan
Hesse, Max Sonnleitner and Gerhard J. Schutz
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.