4-Cyanoindole-2′-deoxyribonucleoside (4CIN): A Universal Fluorescent Nucleoside Analogue

Abstract

The synthesis and characterization of a universal and fluorescent nucleoside, 4-cyanoindole-2'-deoxyribonucleoside (4CIN), and its incorporation into DNA is described. 4CIN is a highly efficient fluorophore with quantum yields >0.90 in water. When incorporated into duplex DNA, 4CIN pairs equivalently with native nucleobases and has uniquely high quantum yields ranging from 0.15 to 0.31 depending on sequence and hybridization contexts, surpassing that of 2-aminopurine, the prototypical nucleoside fluorophore. 4CIN constitutes a new isomorphic nucleoside for diverse applications.

Synthesis of 4‐Cyanoindole Nucleosides, 4‐Cyanoindole‐2ʹ‐Deoxyribonucleoside‐5ʹ‐Triphosphate (4CIN‐TP), and Enzymatic Incorporation of 4CIN‐TP into DNA

“…2AP‐TP has been shown to be efficiently incorporated opposite a template T (Sowers, Fazakerley, Eritja, Kaplan, & Goodman, ; Watanabe & Goodman, ) and this is reflected in the data shown in Table , with K M values 33‐ to 66‐fold weaker for template bases A, C, and G. Here, we show with polymerase primer extension assays that 4CIN‐TP has a similar K M for all template bases (Table ). This correlates well with the previous finding that 4CIN hybridizes non‐discriminately with all bases in a similar thermal range in duplex DNA (Passow & Harki, ). Therefore, we conclude that 4CIN‐TP is a fluorescent nucleotide that can be enzymatically incorporated into DNA across all of the natural templating nucleobases with similar efficiencies.…”

“…While each class has unique uses and properties, our laboratories are most interested in new isomorphic analogues, which are analogues that remain faithful to the size and shape of a naturally occurring nucleobase. In a recent publication, we described the synthesis and extensive characterization of 4CIN, which exhibits strong fluorescence not only as a monomer, but also in a DNA context (Passow & Harki, ). This molecule was subsequently used by Gai and coworkers in a proof of concept study showing that 4CIN could be used to report DNA‐protein binding including the calculation of a binding affinity (Ahmed et al., ).…”

“…Basic Protocol 1 reiterates the published synthesis of 4CIN and its corresponding phosphoramidite (4) for use in automated DNA synthesis (Passow & Harki, 2018). The synthesis route is shown in Figure 1 and starts from commercially available 4-cyanoindole (4CI) and couples this nucleobase to commercial 3,5-di-O-toluoyl-α-1-chloro-2-deoxy-D-ribofuranose (1) to yield 2.…”

“…Calculate the yield of the reaction by dissolving the final product in ddH 2 O. Using Beer's law and the published 4CIN ε 300 = 7790 M −1 cm −1 , calculate a molarity and then moles of product (Passow & Harki, 2018 (d,J = 8.4 Hz,1H),7.86 (d,J = 3.5 Hz,1H),7.65 (d,J = 7.4 Hz,1H),7.41 (t,J = 7.9 Hz,1H),6.87 (d,J = 3.4 Hz,1H),6.62 (dd,J = 8.3,6.1 Hz,1H),1H),2H),3.19 (q,J = 7.3 Hz,18H),2.84 (ddd,J = 14.3,8.3,6.3 Hz,1H),2.49 (ddd,J = 14.0,6.1,2.9 Hz,1H),1.28 (t,J = 7.3 Hz,26H).…”

“…Fluorescent nucleosides are an ever growing class of molecules with diverse biological applications (Sinkeldam, Greco, & Tor, 2010;Xu, Chan, & Kool, 2017 the unique spectral properties and environmental sensitivities of fluorescent nucleosides, a variety of fundamental studies involving polymerases (Joyce et al, 2008), base-flipping enzymes (Kilin et al, 2017), and processes associated with DNA damage (Wilson, Beharry, Srivastava, O'Connor, & Kool, 2018) have been achieved. This article provides detailed synthesis procedures for four different nucleoside and nucleotide molecules based on the fluorophore 4-cyanoindole (4CI), including the recently published 4-cyanoindole-2ʹ-deoxyribonucleoside (4CIN) and its phosphoramidite (Passow & Harki, 2018), as well as new analogues 4-cyanoindole-ribonucleoside (4CINr) and the 5ʹ-triphosphate of 4CIN, 4CIN-TP. The article ends with assay protocols to evaluate the enzymatic incorporation of 4CIN-TP into DNA using a DNA polymerase.…”