Fluorescent RNA cytosine analogue – an internal probe for detailed structure and dynamics investigations

Abstract The bright fluorescent cytosine analogue tCO stands out among fluorescent bases due to its virtually unquenched fluorescence emission in duplex DNA. However, like most reported base analogues, it has not been thoroughly characterized in RNA. We here report on the first synthesis and RNA-inc...

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Autores principales:	Anders Foller Füchtbauer, Søren Preus, Karl Börjesson, Scott A. McPhee, David M. J. Lilley, L. Marcus Wilhelmsson
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f9924c7b2eb845ad804425dc82e6bff6
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spelling	oai:doaj.org-article:f9924c7b2eb845ad804425dc82e6bff62021-12-02T16:06:37ZFluorescent RNA cytosine analogue – an internal probe for detailed structure and dynamics investigations10.1038/s41598-017-02453-12045-2322https://doaj.org/article/f9924c7b2eb845ad804425dc82e6bff62017-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-02453-1https://doaj.org/toc/2045-2322Abstract The bright fluorescent cytosine analogue tCO stands out among fluorescent bases due to its virtually unquenched fluorescence emission in duplex DNA. However, like most reported base analogues, it has not been thoroughly characterized in RNA. We here report on the first synthesis and RNA-incorporation of tCO, and characterize its base-mimicking and fluorescence properties in RNA. As in DNA, we find a high quantum yield inside RNA duplexes (<ΦF> = 0.22) that is virtually unaffected by the neighbouring bases (ΦF = 0.20–0.25), resulting in an average brightness of 1900 M−1 cm−1. The average fluorescence lifetime in RNA duplexes is 4.3 ns and generally two lifetimes are required to fit the exponential decays. Fluorescence properties in ssRNA are defined by a small increase in average quantum yield (<ΦF > = 0.24) compared to dsRNA, with a broader distribution (ΦF = 0.17–0.34) and slightly shorter average lifetimes. Using circular dichroism, we find that the tCO-modified RNA duplexes form regular A-form helices and in UV-melting experiments the stability of the duplexes is only slightly higher than that of the corresponding natural RNA (<ΔT m> = + 2.3 °C). These properties make tCO a highly interesting fluorescent RNA base analogue for detailed FRET-based structural measurements, as a bright internal label in microscopy, and for fluorescence anisotropy measurements of RNA dynamics.Anders Foller FüchtbauerSøren PreusKarl BörjessonScott A. McPheeDavid M. J. LilleyL. Marcus WilhelmssonNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-8 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Anders Foller Füchtbauer Søren Preus Karl Börjesson Scott A. McPhee David M. J. Lilley L. Marcus Wilhelmsson Fluorescent RNA cytosine analogue – an internal probe for detailed structure and dynamics investigations
description	Abstract The bright fluorescent cytosine analogue tCO stands out among fluorescent bases due to its virtually unquenched fluorescence emission in duplex DNA. However, like most reported base analogues, it has not been thoroughly characterized in RNA. We here report on the first synthesis and RNA-incorporation of tCO, and characterize its base-mimicking and fluorescence properties in RNA. As in DNA, we find a high quantum yield inside RNA duplexes (<ΦF> = 0.22) that is virtually unaffected by the neighbouring bases (ΦF = 0.20–0.25), resulting in an average brightness of 1900 M−1 cm−1. The average fluorescence lifetime in RNA duplexes is 4.3 ns and generally two lifetimes are required to fit the exponential decays. Fluorescence properties in ssRNA are defined by a small increase in average quantum yield (<ΦF > = 0.24) compared to dsRNA, with a broader distribution (ΦF = 0.17–0.34) and slightly shorter average lifetimes. Using circular dichroism, we find that the tCO-modified RNA duplexes form regular A-form helices and in UV-melting experiments the stability of the duplexes is only slightly higher than that of the corresponding natural RNA (<ΔT m> = + 2.3 °C). These properties make tCO a highly interesting fluorescent RNA base analogue for detailed FRET-based structural measurements, as a bright internal label in microscopy, and for fluorescence anisotropy measurements of RNA dynamics.
format	article
author	Anders Foller Füchtbauer Søren Preus Karl Börjesson Scott A. McPhee David M. J. Lilley L. Marcus Wilhelmsson
author_facet	Anders Foller Füchtbauer Søren Preus Karl Börjesson Scott A. McPhee David M. J. Lilley L. Marcus Wilhelmsson
author_sort	Anders Foller Füchtbauer
title	Fluorescent RNA cytosine analogue – an internal probe for detailed structure and dynamics investigations
title_short	Fluorescent RNA cytosine analogue – an internal probe for detailed structure and dynamics investigations
title_full	Fluorescent RNA cytosine analogue – an internal probe for detailed structure and dynamics investigations
title_fullStr	Fluorescent RNA cytosine analogue – an internal probe for detailed structure and dynamics investigations
title_full_unstemmed	Fluorescent RNA cytosine analogue – an internal probe for detailed structure and dynamics investigations
title_sort	fluorescent rna cytosine analogue – an internal probe for detailed structure and dynamics investigations
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/f9924c7b2eb845ad804425dc82e6bff6
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Fluorescent RNA cytosine analogue – an internal probe for detailed structure and dynamics investigations

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