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

Anders Foller Füchtbauer, Søren Preus, Karl Börjesson, Scott A. McPhee, David M. J. Lilley, L. Marcus Wilhelmsson (Lead / Corresponding author)

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Abstract

The bright fluorescent cytosine analogue tC(O) 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 tC(O), 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 tC(O)-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 tC(O) 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.

Original languageEnglish
Article number2393
Pages (from-to)1-8
Number of pages8
JournalScientific Reports
Volume7
DOIs
Publication statusPublished - 24 May 2017

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Cytosine
RNA
Fluorescence
Fluorescence Polarization
DNA
Circular Dichroism
Freezing
Microscopy

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Füchtbauer, Anders Foller ; Preus, Søren ; Börjesson, Karl ; McPhee, Scott A. ; Lilley, David M. J. ; Wilhelmsson, L. Marcus. / Fluorescent RNA cytosine analogue - an internal probe for detailed structure and dynamics investigations. In: Scientific Reports. 2017 ; Vol. 7. pp. 1-8.
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abstract = "The bright fluorescent cytosine analogue tC(O) 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 tC(O), 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 tC(O)-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 tC(O) 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.",
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Fluorescent RNA cytosine analogue - an internal probe for detailed structure and dynamics investigations. / Füchtbauer, Anders Foller; Preus, Søren; Börjesson, Karl; McPhee, Scott A.; Lilley, David M. J.; Wilhelmsson, L. Marcus (Lead / Corresponding author).

In: Scientific Reports, Vol. 7, 2393, 24.05.2017, p. 1-8.

Research output: Contribution to journalArticle

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T1 - Fluorescent RNA cytosine analogue - an internal probe for detailed structure and dynamics investigations

AU - Füchtbauer, Anders Foller

AU - Preus, Søren

AU - Börjesson, Karl

AU - McPhee, Scott A.

AU - Lilley, David M. J.

AU - Wilhelmsson, L. Marcus

N1 - The authors thank the Swedish Research Council (VR) [2008-4990 to L.M.W.], Olle Engkvist Byggmästare Foundation (L.M.W.) and Cancer Research UK (D.M.J.L.) for financial support

PY - 2017/5/24

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N2 - The bright fluorescent cytosine analogue tC(O) 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 tC(O), 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 tC(O)-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 tC(O) 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.

AB - The bright fluorescent cytosine analogue tC(O) 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 tC(O), 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 tC(O)-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 tC(O) 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.

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DO - 10.1038/s41598-017-02453-1

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JO - Scientific Reports

JF - Scientific Reports

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