Introduction

Fluorescence resonance energy transfer (FRET) is a powerful technique for studying nucleic acids (1-3). Typically, two fluorophores are employed in a FRET experiment, a donor and an acceptor. To allow energy transfer the fluorescence spectrum of the donor fluorophore should overlap with the absorption spectrum of the acceptor fluorophore. The efficiency of energy transfer is

From: Methods in Molecular Biology, vol. 335: Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols Edited by: V. V. Didenko © Humana Press Inc., Totowa, NJ

strongly dependent on the distance between the two fluorophores. Therefore, FRET has been used as a "molecular ruler" to probe molecular interactions at a distance between 10 and 100 A. With the advance of solid-phase nucleic acids synthesis and the availability of a broad range of fluorophores with different absorption and emission properties, multi-fluorophore FRET is emerging as a new technique for monitoring the structure and dynamics of complicated biomolecules (4-11). In a multi-fluorophore FRET experiment, a different fluorophore is attached to each site of interest in the biomolecule with more than two branches. If any two among the different fluorophore labels can form a FRET pair with efficient energy transfer, the FRET efficiency and distance information between each of the pairs can be obtained in a single experiment.

Multi-fluorophore FRET has several advantages over dual-fluorophore FRET for studying complicated biomolecules. A multi-fluorophore system decreases the number of experiments. For example, to study a three-way DNA junction, three dual-fluorophore FRET experiments would be required. In comparison, only one tri-fluorophore FRET experiment is needed to obtain the same information. More importantly, because all data are collected in a single system for multi-fluorophore FRET experiments, the results should be more consistent and less prone to errors than those from several experiments.

Because of these advantages, multi-fluorophore FRET has become an important tool for studying nucleic acids. For example, folding of a tri-fluorophore-labeled DNAzyme has been measured by this method, from which a Zn2+-dependent two-step folding mechanism has been identified (6). A study of ribosomal protein S15 binding to a three-way DNA junction has also been carried out using the tri-fluorophore FRET method (9), in which two fluorophores were attached to the DNA junction and a third to the ribosomal protein. As a result, the binding kinetics as well as the accompanying structural change of the DNA junction was elucidated simultaneously.

A number of methods are available for quantitative FRET measurement for dual-labeled systems, including monitoring of fluorescence intensity or lifetime of the donor, or the emission of the sensitized acceptor (2). There are several advantages to monitoring the sensitized acceptor emission with a technique known as the (ratio)A method (see Subheading 3.3.2. for the dual-fluorophore (ratio)A method). First, all measurements are taken in a single solution, yielding results with high consistency. Second, the quantum yield of the acceptor fluorophore does not affect the calculation. Finally, the concentration of the sample is not important so long as a sufficient signal can be obtained (2). In this chapter, we wish to take FRET measurements one step further and apply the (ratio)A method to multi-fluorophore systems. As an example, FRET study of a tri-fluorophore-labeled DNAzyme is presented to illustrate the experimental details for application of multi-fluorophore FRET. The DNAzyme

Substrate strand

3'-GTAGAGAAGGATAT CACT CA -5' -FAM Cy5-5'-C ATCTCTTCT ATAGTGAGT -3'

Fig. 1. The secondary structure of the tri-fluorophore-labeled "8-17" DNAzyme showing positions of the three fluorophores: FAM, TMR, and Cy5. The substrate strand shown in the figure is not the native substrate. The base with an underline is a ribo-adenosine in the native substrate, which is changed to a deoxyribo-adenosine for fluorescence resonance energy transfer studies to prevent metal-induced cleavage.

studied in the experiment, obtained through in vitro selection, is known as the "8-17" DNAzyme, whose secondary structure is shown in Fig. 1 (12-14). The DNAzyme contains a substrate strand and an enzyme strand. In its active form, the substrate strand contains a single ribonucleotide linkage in the middle of deoxyribonucleotides, which can be cleaved in the presence of divalent metal ions (see the caption of Fig. 1). For the FRET study, the substrate strand consists of an all-deoxyribonucleotide analog to prevent metal-induced cleavage during the study. A FAM is labeled at the 5'-end of the substrate, whereas a Cy5 is labeled at the 5'-end of the enzyme. To probe the branched arm in the DNAzyme, an internal 5-carboxyltetramethylrhodamin (TMR) is labeled at the 16th cytosine base of the enzyme strand (6).

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