PREFACE

Fluorescence spectroscopy, an established and highly sensitive analytical technique, has been extensively used by the scientific community for many years. For decades, however, the majority of users have relied on a limited number of established fluorophores, either naturally occurring or of synthetic origin. This has dramatically changed in recent years.

Major technological advances in fluorescence-based instrumentation and techniques, including single-molecule spectroscopy, have triggered a renewed interest in the synthesis and development of new fluorescent probes and labels. Two major paths have been taken that are fundamentally related to the above-mentioned two (i.e., of biosynthetic or synthetic origin) but differ in their accommodation of the challenges presented by modern techniques and contemporary scientific questions. A particularly intriguing and emerging area of research, which is highlighted in this book, is the fabrication of minimally perturbing fluorescent analogs of otherwise nonemissive biological building blocks, including amino acids, lipids, and nucleosides.

To share with the reader the renaissance in this field of fluorescent biomolecules and their building blocks, we open with a general and concise tutorial of fluorescence spectroscopy. As readers would appreciate, it is practically impossible to capture all the nuances associated with the development of new fluorescent probes in such a book. To partially correct for this "deficiency," the second chapter provides a condensed overview of naturally occurring and synthetic fluorescent biomolecular building blocks, addressing the core issues and key advances in this field. Selected topics are then elaborated on in individual chapters.

While most laboratories utilize steady state and perhaps basic time-resolved techniques, a great deal of information can be obtained from more sophisticated experiments. Albinsson and Nordén discuss the theory and applications of polarized light spectroscopy-based techniques and their application for the study of biomolecules. Such experiments can be done in bulk solution as well as in microscopy and single-molecule modalities to provide information about the separation and orientation of chromophores.

Before moving on to discuss new synthetic chromophores in later chapters, we first cover fluorescent proteins as they have become the cornerstone of modern biophysics. Two main approaches are typically considered. One relies on the genetic expression of the classical green fluorescent protein and its variants, where the chromophore is generated from the spontaneous condensation of naturally occurring amino acids as discussed by Jung. A distinct approach, presented by Durkin and Budisa, relies on the incorporation of intrinsically fluorescent noncanonical amino acids by in vitro translation techniques, which exploit an expanded genetic code. Both techniques are extremely powerful and provide experimentalists with an enhanced toolbox of emissive proteins, but rely on rather sophisticated biochemical techniques for protein expression. A simplified approach is discussed by Armitage, where genetically encoded antibody fragments and fluorogenic dyes assemble noncovalently to form bright fluorescent complexes.

One element, distinguishing protein biochemists from the community interested in nucleic acids is that, unlike aromatic amino acids that are emissive, the canonical DNA and RNA nucleosides are all practically nonemissive. This has triggered rather extensive efforts aimed at the synthesis and implementation of fluorescent nucleoside analogs. Several approaches are covered here. Saito and Bag discuss diverse families of solvatochromic nucleosides produced by either covalently linking known chromophores to the native nucleosides or by conjugating additional aromatic rings to the native nucleobases. Chicas and Hudson specifically discuss fluorescent cytidine analogs, with emphasis on pyrrolo-C and its derivatives, both in the context of oligonucleotides and in PNAs. Sekine and coworkers elaborate on another family of pyrimidine analogs built around the pyrimidopyrimidoindole motif. While diverse applications have previously been reported, the authors focus here on the implementation of this responsive family of emissive C analogs within triple-stranded motifs. In contrast to the responsive families of fluorescent C analogs mentioned above, Wilhelmsson describes a family of minimally responsive chromophores, which makes them ideal for FRET studies. Well-matched FRET pairs, unique among nucleoside analogs, can then be used to accurately assess nucleobase-nucleobase distance and orientation, generating high-resolution 3-D structural information.

Although the birth of fluorescent nucleoside analogs as a field is frequently attributed to Stryer's 1969 disclosure of 2-aminopurine, an archetypical and extensively employed emissive nucleoside, the number of newly developed and useful purine analogs is substantially smaller compared to their pyrimidine counterparts. This is partially due to synthetic considerations but also likely reflects that modifying the purine core, unlike that of the pyrimidines, frequently hampers their WC and Hoogsteen pairing abilities as well their accommodation within higher structures. In this context, Luedtke describes useful 8-modified purine analogs, which are exploited for the study of G-quadruplexes without detrimental structural effects. Sinkeldam and Tor then discuss the design and implementation of minimally perturbing yet responsive fluorescent nucleoside analogs, frequently referred to as isomorphic surrogates. Structural and functional elements imparting sensitivity to environmental factors (such as polarity, viscosity, and pH) are introduced into the nucleosidic skeleton with the smallest possible size and functional perturbation.

While all analogs described were designed to form WC pairs and be paired with their native complementary nucleobases, Hirao and coworkers discuss unnatural base pair systems, where both partners selectively recognize one another and discriminate against the canonical nucleobases. While some of the analogs made are in fact emissive, such selective pairing practically expands the genetic code and facilitates the incorporation of other bright fluorescent labels with high efficiency and selectivity. Deviating even further from the canonical structure of the native nucleosides, Crisalli and Kool replace the native heterocyclic nucleobases with aromatic fluorophores, while maintaining the phosphate-sugar backbone. Due to their chromophore-chromophore interactions, such DNA-like oligomers, coined fluorosides, display unique photophysical features and provide a fertile motif for the combinatorial discovery of new sensors and labels.

Similarly to the biomolecular building blocks of proteins and nucleic acids, the majority of membrane components are nonemissive. Designing emissive analogs to study these unique assemblies imposes certain structural and functional issues. Chattopadhyay and colleagues review several popular membrane probes and highlight their potential for extracting information on the environment, organization, and dynamics of membranes. Cebecauer and Sachl then take a rather comprehensive look at diverse fluorescent probes that have been developed to assess lipid phases and their separation, membrane viscosity, and curvature as well as pH and potential. They conclude by discussing future directions and cell biology questions that may be addressed in future using lipophilic fluorescent probes.

We conclude this book with a rather unique chapter discussing small fluorophores that don't serve as components of higher molecular weight biomolecules or assemblies. Wilson discusses the design and utility of fluorescent neurotransmitter analogs as tools for exploring neurotransmission and its regulation. Such analogs can be used to investigate receptors, enzymes, and transporters that interact with native neurotransmitters.

As most readers appreciate, contemporary fluorescence spectroscopy, with all its experimental variations, touches numerous and very diverse fields. Yet, with all the technological advances, in its most fundamental level, this amazing spectroscopy relies on the availability of suitably designed fluorescent probes. The creative and elegant approaches presented here highlight how judiciously designed and implemented fluorescence probes could significantly promote advances in biophysics, biochemistry, and structural biology. What is perhaps less obvious is that the design and implementation of such probes remains an empirical exercise. Our ability to predict the intricate photophysical features of designer probes and their response to diverse environmental effects is still rather primitive and, for the most part, qualitative. It is likely (and it is certainly our hope) that computational approaches developed in coming years will refine the experimentalists' approach, which frequently relies on trial and error. Nevertheless, as evidenced by two Nobel prizes awarded in recent years (R. Y. Tsien, M. Chalfie, and O. Shimomura in 2008 and W. E. Moerner, S. W. Hell, and E. Betzig in 2014), fluorescence spectroscopy continues to pave the road forward in critical scientific disciplines. We hope that this book inspires the next generation of young scientists to dive into this fascinating field and spend their creative years ensuring that the future of this field remains bright and colorful!

Assembling such a collection of quality chapters, as any editor knows, takes far longer than originally expected and planned. It requires the ultimate cooperation of authors, reviewers, and publishers. We...