Plug-and-play fluorophores extend the spectral properties of spinach

Article abstract of DOI:10.1021/ja410819x

Spinach and Spinach2 are RNA aptamers that can be used for the genetic encoding of fluorescent RNA. Spinach2 binds and activates the fluorescence of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (DFHBI), allowing the dynamic localizations of Spinach2-tagged RNAs to be imaged in live cells. The spectral properties of Spinach2 are limited by DFHBI, which produces fluorescence that is bluish-green and is not optimized for filters commonly used in fluorescence microscopes. Here we characterize the structural features that are required for fluorophore binding to Spinach2 and describe novel fluorophores that bind and are switched to a fluorescent state by Spinach2. These diverse Spinach2-fluorophore complexes exhibit fluorescence that is more compatible with existing microscopy filter sets and allows Spinach2-tagged constructs to be imaged with either GFP or YFP filter cubes. Thus, these plug-and-play fluorophores allow the spectral properties of Spinach2 to be altered on the basis of the specific spectral needs of the experiment.

Full text of DOI:10.1021/ja410819x

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Communication
Plug-and-play fluorophores extend the spectral properties of Spinach
Wenjiao Song, Rita L Strack, Nina Svensen, and Samie R. Jaffrey
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 06 Jan 2014
Downloaded from http://pubs.acs.org on January 7, 2014
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Plug-and-play fluorophores extend the spectral properties of
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Wenjiao Song, Rita L. Strack, Nina Svensen, Samie R. Jaffrey*
Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10065, USA.
Supporting Information Placeholder
Furthermore, experiments are needed to confirm that the function
of the target RNA is not inhibited by the aptamer. In the case of
5S, 7SK, CGGꢀrepeat toxic RNAs and various bacterial mRNAs,
insertion sites have already been identified that tolerate Spinach
and/or Spinach2.5ꢀ7 These RNAs interact with (Z)ꢀ4ꢀ(3,5ꢀ
difluoroꢀ4ꢀhydroxybenzylidene)ꢀ1,2ꢀdimethylꢀ1Hꢀimidazolꢀ
5(4H)ꢀone (DFHBI) to produce a bluishꢀgreen fluorescence emisꢀ
sion (501 nm) after excitation at 447 nm. Because of the comꢀ
plexity of finding an insertion site in an RNA for Spinach, it
would be desirable to not have to reoptimize the RNA for a difꢀ
ferent aptamer tag in order to perform experiments requiring difꢀ
ferent fluorescence excitation and/or emission properties.
ABSTRACT: Spinach and Spinach2 are RNA aptamers that can
be used for the genetic encoding of fluorescent RNA. Spinach2
binds and activates the fluorescence of (Z)ꢀ4ꢀ(3,5ꢀdifluoroꢀ4ꢀ
hydroxybenzylidene)ꢀ1,2ꢀdimethylꢀ1Hꢀimidazolꢀ5(4H)ꢀone
(DFHBI), allowing the dynamic localizations of Spinach2ꢀtagged
RNAs to be imaged in live cells. The spectral properties of
Spinach2 are limited by DFHBI, which produces fluorescence
that is bluishꢀgreen and is not optimized for filters commonly
used in fluorescence microscopes. Here we characterize the
structural features that are required for fluorophore binding to
Spinach2 and describe novel fluorophores that bind and are
switched to a fluorescent state by Spinach2. These diverse
Spinach2ꢀfluorophore complexes exhibit fluorescence that is
more compatible with existing microscopy filter sets and allows
Spinach2ꢀtagged constructs to be imaged with either GFP or YFP
filter cubes. Thus, these “plugꢀandꢀplay” fluorophores allow the
spectral properties of Spinach2 to be altered based on the specific
spectral needs of the experiment.
A limitation of Spinach2 is that it has suboptimal spectral
characteristics for fluorescence imaging using standard widefield
microscopes. Spinach and Spinach2 bound to DFHBI have fluoꢀ
rescence excitation maxima of 447 nm, and peak fluorescence
emission of 501 nm.5ꢀ6 These wavelengths do not fully match the
filter cubes used for imaging green fluorescence on most microꢀ
scopes. Typically, these are optimized to detect GFP or fluoresꢀ
cein isothiocyanate, and have a bandpass excitation filter that
transmits light at 480 nm +/ꢀ 20 nm, a dichroic mirror set at 505
nm, and an emission filter that transmits light at 535 nm +/ꢀ 20
nM. As a result, Spinach2ꢀDFHBI complexes are exposed to
excitation light that does not lead to maximum fluorescence.
Additionally, a considerable portion of the emitted light from
Spinach2ꢀDFHBI is not collected since it is blocked by the diꢀ
chroic mirror or emission filter.
Imaging RNA in living cells is important for understanding
the function and regulation of diverse classes of cellular RNAs.
A strategy for imaging RNAs is to express RNAs with sequence
tags that confer fluorescence to the RNA.1 These tags include
sequences that recruit GFP.2 A related approach involves the use
of two sequence tags to template the formation of GFP from each
half of split GFP.3 An alternate strategy is to use RNA sequences
that exhibit fluorescence upon binding small molecules. Several
RNA aptamers that bind and switch on the fluorescence of variꢀ
ous small molecule dyes have been described.4 The use of these
dyes is limited because their fluorescence is nonspecifically actiꢀ
vated by cellular components.5 A recent approach to overcome
this problem uses RNA aptamers that bind and induce the fluoresꢀ
cence fluorophores resembling those in GFP.5 The brightest of
these RNAꢀfluorophore complexes are Spinach and a recently
improved variant, Spinach2, which exhibits improved folding and
thermostability.5ꢀ6 Because these fluorophores exhibit low backꢀ
ground fluorescence when incubated with cells, the dynamics of
RNA localization in cells can be imaged by engineering cells to
express Spinach2 fused to target RNA molecules of interest.5ꢀ6
Although we have described other RNAꢀfluorophore comꢀ
plexes with different excitation and emission maxima, these are
not as bright and not optimized to have the same efficient folding
as Spinach2.5ꢀ6 Thus, improved and/or novel spectral properties
of Spinach2ꢀfluorophore complexes could significantly enhance
RNA imaging.
We considered the possibility that modifications to DFHBI
could redꢀshift the excitation and emission properties of the Spinꢀ
ach2ꢀfluorophore complex. To test this idea, we first sought to
understand the structural features of DFHBI that are required for
Spinach2 to activate its fluorescence. We first examined the role
of substituents on the benzylidene ring. Spinach2 tolerated fluorꢀ
ophores containing different halogens in place of fluorine. For
example, switching the fluorines to either bromine ((Z)ꢀ4ꢀ(3,5ꢀ
dibromoꢀ4ꢀhydroxyꢀbenzylidene)ꢀ1,2ꢀdimethylꢀ1Hꢀimidazolꢀ
Tagging an RNA with Spinach for fluorescent imaging reꢀ
quires identification of an insertion site in the target RNA that is
compatible with Spinach folding. Spinach folding can be inhibitꢀ
ed by neighboring flanking sequences, presumably due to hybridꢀ
ization interactions that prevent Spinach folding.6 Researchers
may therefore need to screen multiple insertion sites in the target
RNA to identify one in which the aptamer can fold efficiently.
5(4H)ꢀone)
or
chlorine
(Z)ꢀ4ꢀ(3,5ꢀdichloroꢀ4ꢀ
hydroxybenzylidene)ꢀ1,2ꢀdimethylꢀ1Hꢀimidazolꢀ5
resulted in compounds that bound to Spinach2 and
(4H)ꢀone)
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Table 1. Photophysical and binding properties of fluorophore-Spinach2 complexes
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Excitation
maximum
Extinction coeffi-
cient
Emission
maximum
(nm)
Fluorescence
quantum yield
KD
(nM)
Bright-
nessb
Fluorophore
(nm)
423
447
(M-1cm-1)a
DFHBI
489
501
30,100
0.0007
0.72
ꢀ
0.13
100
Spinach2ꢀDFHBI
22,000
530
9
DFHBIꢀ1T
Spinach2ꢀDFHBIꢀ1T
DFHBIꢀ2T
426
482
460
495
505
515
35,400
31,000
34,800
0.00098
0.94
ꢀ
560
ꢀ
0.22
184
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ꢀ
0.0012
0.26
Spinach2ꢀDFHBIꢀ2T
500
523
29,000
0.12
1300
22
aExtinction coefficients were all measured at pH7.4. bBrightness (extinction coefficient × quantum yield) is reported relative to Spinach2ꢀ
DFHBI.
Figure 1. Different fluorophores bind Spinach2 and form RNAꢀfluorophore complexes with unique spectral properties. Excitation (dotted
line) and emission (solid line) spectra of Spinach2ꢀfluorophore complexes. Excitation and emission spectra were collected in the presence
of excess Spinach2 (5 ꢁM) in the presence of different fluorophores (1 ꢁM) in binding buffer (40 mM HEPES, pH 7.4, 125 mM KCl, 10
mM MgCl2): (a) DFHBI, (b) DFHBIꢀ1T, and (c) DFHBIꢀ2T. Spectra are shown as the percent of the maximum excitation and emission
fluorescence for each Spinach2ꢀfluorophore complex. Excitation spectra were collected at the indicated emission maximum wavelength,
and emission spectra were collected at the indicated excitationꢀmaximum wavelength. The numbering in (a) indicates the atom numbering
scheme for the imidazolone ring.
exhibited only a slight reduction in overall fluorescence intensity
compared to DFHBI (Table S1). However, neither of these comꢀ
pounds showed a substantial shift in the peak excitation or emisꢀ
sion wavelength when bound to Spinach2 (Table S1, Figure S1).
GFPꢀlike fluorophores is markedly influenced as the electron
distribution is shifted towards the Nꢀ1 and Cꢀ2 position.4 The
HOMOꢀLUMO gap directly influences the excitation and emisꢀ
sion wavelengths of the fluorophore. We therefore tested the
effects of various electronꢀwithdrawing substituents on both posiꢀ
tions.
We next examined if removing one fluorine would result in
redꢀshifted fluorescence. However Spinach2 bound to ((Z)ꢀ4ꢀ(3ꢀ
fluoroꢀ4ꢀhydroxybenzylidene)ꢀ1,2ꢀdimethylꢀ1Hꢀimidazolꢀ5(4H)ꢀ
one) again exhibited only a negligible red shift in both the excitaꢀ
tion or emission maxima as well as a slight reduction in fluoresꢀ
cence because of the increased pKa, which reduces the amount of
fluorophore in the higher fluorescence phenolate form (Figure
S2). Taken together, these data indicate that fluorophores with
altered halogen substituents on the benzylidene ring bind Spinꢀ
ach2 but do not exhibit markedly shifted spectral properties. Preꢀ
vious studies have shown that the HOMOꢀLUMO energy gap for
We first examined the effects of substitutions at the Nꢀ1 poꢀ
sition of the imidazolone ring. Replacing the methyl substituent
in DFHBI with a 1,1,1ꢀtrifluoroethyl substituent (DFHBIꢀ1T, (Z)ꢀ
4ꢀ(3,5ꢀdifluoroꢀ4ꢀhydroxybenzylidene)ꢀ2ꢀmethylꢀ1ꢀ(2,2,2ꢀ
trifluoroethyl) ꢀ1Hꢀimidazolꢀ5(4H)ꢀone) resulted in a Spinach2
complex with a 35 nm red shift in the excitation peak, and a slight
red shift in the emission peak (Figure 1, Table 1). DFHBIꢀ1T
also exhibited an overall increase in brightness, which reflects a
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slight increase in the extinction coefficient and an increase in the
quantum yield (Table 1).
sistent with improved excitation of Spinach2 complexed with
DFHBIꢀ1T.
Additionally, we noticed that cells treated with 20 ꢁM
1
2
We next tested the effect of an aminoethyl substituent at the
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4
5
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7
8
9
Nꢀ1 position (DFHBIꢀ1AE, (Z)ꢀ1ꢀ(2ꢀaminoethyl)ꢀ4ꢀ(3,5ꢀdifluoroꢀ
DFHBIꢀ1T exhibited lower background fluorescence than cells
treated with the same concentration of DFHBI. Typically DFHBIꢀ
4ꢀhydroxybenzylidene)ꢀ2ꢀmethylꢀ1Hꢀimidazolꢀ5(4H)ꢀone).
At
neutral pH, the aminoethyl substituent is protonated, which makes
this substituent electron withdrawing. This molecule exhibited a
30 nm red shift in the excitation and a 9 nm red shift in the emisꢀ
sion (Figure S1, Table S1). The overall brightness of this comꢀ
plex was similar to the brightness of the Spinach2 bound to
DFHBI. Spinach2 bound to DFHBIꢀ1AE exhibited an 18% reꢀ
duction in the extinction coefficient and a 24% reduction in quanꢀ
tum yield (Table S1).
treated
cells
exhibit
a
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We also tested the effect of switching the Nꢀ1 alkyl substituꢀ
ent to a hydroxyl (DFHBIꢀ1HO, (Z)ꢀ4ꢀ(3,5ꢀdifluoroꢀ4ꢀhydroxyꢀ
benzylidene)ꢀ1ꢀhydroxyꢀ2ꢀmethylꢀ1Hꢀimidazolꢀ5(4H)ꢀone) or a
methoxyl substituent (DFHBIꢀ1MO, (Z)ꢀ4ꢀ(3,5ꢀdifluoroꢀ4ꢀ
hydroxybenzylidene)ꢀ1ꢀmethoxyꢀ2ꢀmethylꢀ1Hꢀimidazolꢀ5(4H)ꢀ
one). DFHBIꢀ1HO exhibited markedly reduced binding to Spinꢀ
ach2, as well as negligible Spinach2ꢀinduced fluorescence (Table
S1). On the other hand, DFHBIꢀ1MO exhibited only a slightly
reduced binding affinity to Spinach2 (Table S1). Spinachꢀ
DFHBIꢀ1MO complexes were fluorescent, but did not exhibit
substantial redꢀshifted emission. The inability DFHBIꢀ1HO to
bind Spinach2 may reflect intramolecular hydrogenꢀbonding inꢀ
teractions between the 1ꢀhydroxyl proton and the ketone oxygen.
This may compete with interactions required for Spinach2 bindꢀ
ing.
Taken together, these results suggest that Nꢀ1 alkyl substituꢀ
ents in the DFHBI core structure are generally well tolerated for
Spinach2 binding. However, only Nꢀ1 trifluoroethyl and amiꢀ
noethyl substituents caused redꢀshifted excitation and emission
spectra.
We also considered the effects of switching the methyl subꢀ
stituent at the Cꢀ2 position of the imidazolinone ring to a trifluoꢀ
romethyl
(DFHBIꢀ2T,
(Z)ꢀ4ꢀ(3,5ꢀdifluoroꢀ4ꢀ
hydroxybenzylidene)ꢀ1ꢀmethylꢀ2ꢀ(trifluoromethyl)ꢀ1Hꢀimidazolꢀ
5(4H)ꢀone). When bound to Spinach2, this molecule exhibited a
marked 53 nm red shift in the excitation and a 22 nm red shift in
the emission maxima, although the overall brightness was someꢀ
what reduced due to a decrease in the quantum yield (Figure 1,
Table 1). The increase in KD to ~1.2 µM (Figure 1, Table 1) sugꢀ
gests that the bulky trifluoromethyl moiety may exhibit steric
hindrance with the Spinach2 aptamer.
Figure 2. Liveꢀcell imaging of Spinach2 fusion RNAs with difꢀ
ferent fluorophores. (a) COS7 cells expressing (CGG)60ꢀSpinach2
in the presence of either DFHBI or DFHBIꢀ1T. Cells were initialꢀ
ly cultured in the presence of 20 ꢁM DFHBI (top panel) and imꢀ
ages were acquired using a 100 msec exposure. The media was
then exchanged with media containing 20 ꢁM DFHBIꢀ1T for 10
min, and images of the same cell nuclei were obtained using idenꢀ
tical image acquisition conditions. Increased fluorescence was
seen in cells cultured with DFHBIꢀ1T (lower panel). (b) Quantifiꢀ
cation of average brightness of 10 foci normalized to brightness
of DFHBI. Average and s.e.m. values are shown. (c) Cells exꢀ
pressing (CGG)60ꢀSpinach2 in the presence of either DFHBI or
DFHBIꢀ2T were imaged using both GFP and YFP filter sets.
Scale bar, 10 ꢁm.
Spinach can be engineered to form RNAs that detect small
molecules in vitro.8 In this approach, Spinach is fused to apꢀ
tamers that bind to small molecules. This converts Spinach from
a constitutively fluorescent complex to a fluorescent species that
is allosterically regulated by small molecule binding. 8a We asked
if DFHBIꢀ1T, and DFHBIꢀ2T enable fluorescence detection in
vitro at different wavelengths than DFHBI. Indeed, incubation of
the Spinach2ꢀbased Sꢀadenosylꢀmethionine (SAM) sensor, comꢀ
posed of Spinach2 and the SAM aptamer, with each of these
fluorophores resulted in SAMꢀdependent fluorescence emissions
ranging from 501 to 523 nm (Figure S3).
diffuse lowꢀlevel fluorescence, even in the absence of Spinach
expression (Figure S4a,b). This fluorescence derives from the
low fluorescence level of DFHBI in the media as well as faint
fluorescence in cells cultured in DFHBIꢀcontaining media. Addiꢀ
tionally, there are occasionally small fluorescent puncta in cells
which reflect interactions of DFHBI with intracellular debrisꢀlike
structures (Figure S4a,b). We suspect that DFHBI is weakly and
nonspecifically activated by certain intracellular components.
This background fluorescence is noticeable when long imaging
times are used, such as when 5SꢀSpinach localization is imaged in
cells.5 Therefore, to image 5SꢀSpinach using DFHBI, the specifꢀ
ic signal is determined by computationally subtracting the empiriꢀ
cally measured background fluorescence, as determined by measꢀ
uring the signal in cellꢀfree portions of the image (Figure S4c).
However, in DFHBIꢀ1Tꢀtreated cells, the 5SꢀSpinach2 signal was
readily detectable without background subtraction due to an inꢀ
We next asked if DFHBIꢀ1T and DFHBIꢀ2T can be used to
image Spinach2ꢀtagged RNA in living cells. For these experiꢀ
ments, we expressed the CGG repeat toxic RNA that causes fragꢀ
ile X ataxia and tremor syndrome.6 This RNA comprises 60 CGG
repeats, and forms mobile intranuclear RNA inclusions in cells.
This RNA was tagged with Spinach2 as described previously.6
Cells were initially treated with media containing 20 µM DFHBI,
resulting in the expected green intranuclear foci, which were imꢀ
aged using a GFP filter cube. However, replacing the media with
media containing 20 µM DFHBIꢀ1T resulted in foci that were
~60% brighter (Figure 2b). The increase in fluorescence is conꢀ
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crease in the specific fluorescence signal, and because the backꢀ
Thus, the spectral properties of Spinach2 can be matched to the
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ground fluorescence was significantly lower (Figure S4).
DFHBIꢀ1T appears to exhibit reduced nonspecific fluorescence
activation by cells and in the media compared to DFHBI. Thus,
Spinach2 exhibits both higher specific fluorescence and lower
background fluorescence when imaged using DFHBIꢀ1T.
specific spectral needs of the experiment. The design of Spinꢀ
ach2ꢀcompatible fluorophores that exhibit farther redꢀshifted
emissions when bound to Spinach2 is a current direction for our
laboratory.
ASSOCIATED CONTENT
Supporting Information
We next sought to characterize the properties of Spinach2
bound to DFHBIꢀ2T in living cells. The spectral properties of
this complex does not overlap with the standard GFP filter cube,
but are instead more compatible with YFP filter cubes, which
typically have an excitation bandpass filter transmitting 500 nm
+/ꢀ 10 nm light, a dichroic mirror at 515 nm, and an emission
filter that transmits 535 nm +/ꢀ 15 nM light. Indeed, cells exꢀ
pressing (CGG)60ꢀSpinach2 exhibited readily detectable intranuꢀ
clear foci when imaged with the GFP filter cube, but only miniꢀ
mal fluorescence when imaged with the YFP filter cube (Figure
2c). However, when the media was switched with media DFHBIꢀ
2T, fluorescence was markedly reduced when imaging with the
GFP filter cube but was readily detectable using the YFP filter
cube (Figure 2c). These data indicate that Spinach2 imaged
DFHBIꢀ2T results in fluorescence that is detectable using the
yellow emission channel.
Materials and methods, details on the synthesis of small moleꢀ
cules, and additional figure and table are included in the supportꢀ
ing information. This material is available free of charge via the
Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
srj2003@med.cornell.edu
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
Our studies point to the importance of substituents on the imꢀ
idazolinone ring for determining the spectral emission properties
of Spinach2ꢀfluorophore complexes. These findings are conꢀ
sistent with previously reported studies that have extensively
derivatized the GFP fluorophore.9 In these experiments, the
fluorophore was markedly influenced by substitutions on the imꢀ
idazolinone ring. This portion of the fluorophore may tolerate
substitutions since the Spinach aptamer was selected on agarose
beads containing DFHBI connected by a linker attached at the Nꢀ
1 position.5 Thus, this position is likely to be highly tolerant of
substitutions without impairing binding to Spinach2.
We thank NMR analytical Core Facility at Memorial Sloanꢀ
Kettering Cancer Center for assistance with high resolution MS,
K. Solntsev for helpful comments, and J. S. Paige for early conꢀ
tributions to this work. This work was supported by NIH grants
to S.R.J. (R01 NS064516 and R01 EB010249) and R.S. (F32
GM106683).
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Tyagi, S. Nat Methods 2009, 6, 331ꢀ8.
Bertrand, E.; Chartrand, P.; Schaefer, M.; Shenoy, S. M.; Singꢀ
An important advance in this study is the identification of
DFHBIꢀ1T. DFHBIꢀ1T exhibits lower background fluorescence
than DFHBI when incubated with cells. Spinach2ꢀDFHBIꢀ1T
exhibits increased fluorescence when imaged with GFP filter
cubes due to improved excitation of the complex, and lower
background fluorescence. Spinach2ꢀDFHBIꢀ1T exhibits a Stokes
shift of 23 nM compared to 54 nm for Spinach2ꢀDFHBI. Altꢀ
hough this smaller Stokes shift in principle requires careful selecꢀ
tion of filter sets for imaging, the excitation and emission spectra
are compatible with filter cubes commonly used to image GFP.
Additionally, DFHBIꢀ2T allows imaging of Spinach2 using the
YFP filter cube. This can be valuable for experiments in which
simultaneous imaging of Spinach2ꢀtagged RNAs and CFPꢀtagged
proteins is desired.
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ACS Synth Biol 2014, in press.
The different fluorophores described here provide a “plugꢀ
andꢀplay” system for RNA imaging in living cells. The ability to
alter the spectral properties of Spinach2 by using different fluoroꢀ
phores provides a versatility that is not seen with fluorescent proꢀ
teins, which typically have fixed fluorescence properties, or in
select cases can switch to a different wavelength. With a single
Spinach2 construct, different spectral properties can be obtained
for fluorescence imaging. Additionally, the imaging properties
can be changed in a single experiment by washing out one fluoroꢀ
phore and adding another. This approach allows Spinach2ꢀtagged
constructs to be imaged with either GFP or YFP filter cubes.
8.
(a) Paige, J. S.; NguyenꢀDuc, T.; Song, W.; Jaffrey, S. R. Sci-
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