Conformational transitions monitored for single molecules in solution.

Abstract

Phenomena that can be observed for a large number of molecules may not be understood if it is not possible to observe the events on the single-molecule level. We measured the fluorescence lifetimes of individual tetramethylrhodamine molecules, linked to an 18-mer deoxyribonucleotide sequence specific for M13 DNA, by time-resolved, singlephoton counting in a confocal fluorescence microscope during Brownian motion in solution. When many molecules were observed, a biexponential fluorescence decay was observed with equal amplitudes. However, on the single-molecule level, the fraction of one of the amplitudes spanned from 0 to unity for a collection of single-molecule detections. Further analysis by fluorescence correlation spectroscopy made on many molecules revealed a process that obeys a stretched exponential relaxation law. These facts, combined with previous evidence of the quenching effect of guanosine on rhodamines, indicate that the tetramethylrhodamine molecule senses conformational transitions as it associates and dissociates to a guanosine-rich area. Thus, our results reveal conformational transitions in a single molecule in solution under conditions that are relevant for biological processes.A new era of molecular analysis was begun by the introduction of extremely sensitive methods to detect and characterize species at the single-molecule level by spectroscopic means. Several techniques to trace individual fluorescent particles in solution have been reported over the last 12 years (1-9). Single-molecule detection (SMD) in solids and on surfaces has also been accomplished (10-14); however, many features important for chemical and biological systems can only be realized in the liquid phase, as is pointed out by reports regarding applications in DNA sequencing (15, 16) or detection and selection of rare events in diagnostics and biotechnology (17). Another important application for the SMD technique is the study of reaction dynamics at the singlemolecule level.Wang and Wolynes (18) have recently reported how measurements of single molecules can make it possible to gain information about phenomena that cannot be easily understood when a large number of molecules are observed simultaneously. A complex behavior (e.g., a distribution of a physical entity) will not provide information on whether all molecules share a common distribution or whether each molecule gives its own specific contribution to the distribution seen for many molecules. Measurements of single molecules can be decidedly important in obtaining such information.Previously (22), but we used it as a sensitive instrument -to provide precise statistical characteristics of the system. Single-Molecule Measurements. To achieve SMD, we measured repeatedly for short periods (t). The ratio of the measurement time to the characteristic passage time of a molecule through the VE is defined as T, the relative measuring time. By selecting only those measurements that experienced a large time integral of the detected fluorescence, we could effectively...

Chapter 12 Using Fluorophore-Labeled Oligonucleotides to Measure Affinities of Protein–DNA Interactions

“…Sometimes the sequence of the oligonucleotide can affect the properties of the attached fluorophore. Time-resolved fluorescence studies of various tetramethylrhodamine (TMR)-or TAMRA-labeled oligonucleotides demonstrate that the fluorophore can have multiple fluorescent lifetimes, suggesting that the probe exists in multiple environments (Edman et al, 1996;Eggeling et al, 1998;Harley et al, 2002;Vamosi et al, 1996). One of these lifetimes may involve an interaction between fluorophore and DNA bases, which can potentially quench fluorescence emission or otherwise affect the measured values (Sauer et al, 1995;Seidel et al, 1996;Sevenich et al, 1998).…”

High-throughput approach for detection of DNA bending and flexibility based on cyclization

“…Although there has been no report of dye-labeling causing a large alteration of DNA structure, a small perturbation could affect the cyclization kinetics. It was shown that TAMRA can stack at DNA ends like an additional base, and fluorescence resonance energy transfer can occur between TAMRA and G nucleotide (30,31). Because no absolute J factors are available from direct cyclization for dye-labeled DNA molecule containing CG cohesive ends due to lack of single bimolecular association constant k 2 , a construct with nonpalindromic CGAT ends was designed (Fig.…”

“…The small perturbation of DNA structure due to dye-labeling may result from the stacking of fluorescein or TAMRA onto the DNA duplex due to hydrophobic and electrostatic interactions (30,31). Because of its positive charge and large aromatic ring, TAMRA might intercalate into DNA base pairs to a small extent.…”

On Death Numbers and Survival Times of Single Dye Molecules

“…[2][3][4] The time is measured during which a single TMR molecule can be observed (survival time) when linked to a 217-bp DNA sequence which itself is bound to a streptavidinized glass surface by a biotin tag at the 5′-end. We observe that the random time after which a molecule dies is exponentially distributed.…”