Topological variants of single-strand DNA (ssDNA) structures, referred to as “functional DNA,” have been detected in regulatory regions of many genes and are thought to affect gene expression. Two fluorescent analogs of deoxycytidine, Pyrrolo-dC (PdC) and 1,3-diaza-2-oxophenoxazine (t ), can be incorporated into DNA. Here, we describe spectroscopic studies of both analogs to determine fluorescent properties that report on structural transitions from double-strand DNA (dsDNA) to ssDNA, a common pathway in the transition to functional DNA structures. We obtained fluorescence-detected circular dichroism (FDCD) spectra, steady-state fluorescence spectra, and fluorescence lifetimes of the fluorophores in DNA. Our results show that PdC is advantageous in fluorescence lifetime studies because of a distinct ~2？ns change between paired and unpaired bases. However, t is a better probe for FDCD experiments that report on the helical structure of DNA surrounding the fluorophore. Both fluorophores provide complementary data to measure DNA structural transitions. 1. Introduction DNA single strands can hybridize to form higher-order functional structures, which include hairpins, triplexes, and quadruplexes [1–4]. The existence and physiological relevance of these secondary structures in vivo have been the subject of much controversy. However, several in vivo techniques have confirmed the presence of DNA secondary structures in telomeres and regulatory regions of specific genes (e.g., BCL-2, c-myc) [5–8]. Secondary structures may also serve as specific targets recognized by drugs, such as actinomycin D and PIPER, as well as transcription factors, such as Sp1, because of their topological variance from duplex Watson-Crick DNA [9, 10]. On account of their occurrence in regulatory regions and their structural peculiarity, functional DNA structures may serve as biological microswitches for altering transcription by silencing or enhancing gene expression [11, 12]. Exercising control over gene expression by controlling the activation of these switches and/or introducing new switches in biological circuits could revolutionize medical research and offer new avenues of treating genetic disorders. For exercising such control, it is imperative to understand the factors affecting the mechanism of formation and maintenance of functional structures at a fundamental level. Numerous fluorescent analogs of DNA bases have been evaluated for examining the subtleties of DNA transitions [13–15]. Locating an appropriate probe that could map the mechanistic aspects of transition of double
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