2-aminopurine as a fluorescent probe of DNA conformation and the DNA–enzyme interface

Abstract

Nearly 50 years since its potential as a fluorescent base analogue was first recognized, 2-aminopurine (2AP) continues to be the most widely used fluorescent probe of DNA structure and the perturbation of that structure by interaction with enzymes and other molecules. In this review, we begin by considering the origin of the dramatic and intriguing difference in photophysical properties between 2AP and its structural isomer, adenine; although 2AP differs from the natural base only in the position of the exocyclic amine group, its fluorescence intensity is one thousand times greater. We then discuss the mechanism of interbase quenching of 2AP fluorescence in DNA, which is the basis of its use as a conformational probe but remains imperfectly understood. There are hundreds of examples in the literature of the use of changes in the fluorescence intensity of 2AP as the basis of assays of conformational change; however, in this review we will consider in detail only a few intensity-based studies. Our primary aim is to highlight the use of time-resolved fluorescence measurements, and the interpretation of fluorescence decay parameters, to explore the structure and dynamics of DNA. We discuss the salient features of the fluorescence decay of 2AP when incorporated in DNA and review the use of decay measurements in studying duplexes, single strands and other structures. We survey the use of 2AP as a probe of DNA-enzyme interaction and enzyme-induced distortion, focusing particularly on its use to study base flipping and the enhanced mechanistic insights that can be gained by a detailed analysis of the decay parameters, rather than merely monitoring changes in fluorescence intensity. Finally we reflect on the merits and shortcomings of 2AP and the prospects for its wider adoption as a fluorescence-decay-based probe.

Photophysical properties of pyrrolocytosine, a cytosine fluorescent base analogue

“…For example, fluorescence of 2APG is quenched by more than 90%. 36 The lifetimes also change by orders of magnitude. The mechanism for this has been investigated extensively.…”

A highly specific sodium aptamer probed by 2-aminopurine for robust Na+sensing

“…The schemes of 2AP pairing with T and C are shown in Figure 3A. We interpret an increased 2AP emission as relaxed base stacking (40,49). Since a stronger 2AP base pair results in a weaker initial fluorescence, this result also supports that the 2AP pairs with T 20 .…”

“…Instead of using techniques that probe global folding of DNA, such as fluorescence resonance energy transfer, studying local folding is more appropriate here. 2-aminopurine (2AP) is a fluorescent adenine analog (40). Its emission intensity is strongly affected by the local base stacking environment (40).…”

“…2-aminopurine (2AP) is a fluorescent adenine analog (40). Its emission intensity is strongly affected by the local base stacking environment (40). Such a property has made 2AP a powerful probe for nucleic acids (40–42), and related analytical applications (43–45).…”

“…Its emission intensity is strongly affected by the local base stacking environment (40). Such a property has made 2AP a powerful probe for nucleic acids (40–42), and related analytical applications (43–45). In this work, we probe the Ce13d using 2AP at a few different positions, allowing us to map its full folding picture.…”