Sequence Requirements of Intrinsically Fluorescent G

Article Cite This: Biochemistry 2018, 57, 4052−4062

pubs.acs.org/biochemistry

Sequence Requirements of Intrinsically Fluorescent G‑Quadruplexes Tat’ána Majerová,† Tereza Streckerová,†,‡ Lucie Bednárová,† and Edward A. Curtis*,† †

The Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague 166 10, Czech Republic Department of Biochemistry and Microbiology, University of Chemistry and Technology, Prague 166 10, Czech Republic

‡

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S Supporting Information *

ABSTRACT: G-Quadruplexes are four-stranded nucleic acid structures typically stabilized by GGGG tetrads. These structures are intrinsically fluorescent, which expands the known scope of nucleic acid function and raises the possibility that they could eventually be used as signaling components in label-free sensors constructed from DNA or RNA. In this study, we systematically investigated the effects of mutations in tetrads, loops, and overhanging nucleotides on the fluorescence intensity and maximum emission wavelength of >500 sequence variants of a reference DNA G-quadruplex. Some of these mutations modestly increased the fluorescence intensity of this G-quadruplex, while others shifted its maximum emission wavelength. Mutations that increased the fluorescence intensity were distinct from those that increased the maximum emission wavelength, suggesting a trade-off between these two biochemical properties. The fluorescence intensity and maximum emission wavelength were also correlated with multimeric state: the most fluorescent G-quadruplexes were monomers, while those with the highest maximum emission wavelengths typically formed dimeric structures. Oligonucleotides containing multiple G-quadruplexes were in some cases more fluorescent than those containing a single G-quadruplex, although this depended on the length and sequence of the spacer linking the G-quadruplexes. These experiments provide new insights into the properties of fluorescent G-quadruplexes and should aid in the development of improved label-free nucleic acid sensors. bind the small-molecule fluorophore malachite green.10 When bound to these aptamers, the fluorescence intensity of malachite green is enhanced >2000-fold.11 Aptamers that enhance the fluorescence intensity of several other fluorophores have recently been developed, including the spinach aptamer12 and the mango aptamer.13 In some cases, these aptamers can be used as sensors or reporters in the context of living cells.14−16 For example, variants of the spinach aptamer that generate an enhanced fluorescent signal in the presence of specific ligands such as SAM and ADP have been constructed and were used to measure the cellular concentrations of these metabolites in Escherichia coli.14 The structural basis of fluorescence has also been elucidated for the spinach and mango aptamers.17,18 In both cases, a G-quadruplex with an unusual topology in the core of the structure binds the fluorophore using stacking energy and hydrogen bonding. Although motifs such as spinach and mango can be considered to be functional analogues of GFP, the mechanism by which they generate fluorescence is fundamentally different because it requires the presence of an external fluorophore. Enhancement of nucleic acid fluorescence in the absence of an external fluorophore has also been described.19−21 This requires a folded DNA or RNA G-quadruplex structure, and the mechanism of enhancement is probably related to the aromatic nature of the nucleotide building blocks of G-

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nder certain conditions, the hydrozoan jellyfish Aequorea victoria produces fluorescent green light.1 Biochemical fractionation experiments using material prepared from thousands of jellyfish revealed that this fluorescence is produced by a 238-amino acid protein called green fluorescent protein (GFP).2,3 The core of GFP contains three amino acids that cyclize to generate an aromatic chromophore called 4-(phydroxybenzylidene)imidazolidin-5-one (HBI).4,5 In the absence of GFP, this chromophore is not fluorescent, but in the context of a cylindrical cavity created by the three-dimensional fold of the protein, its fluorescence is significantly enhanced.6,7 In addition to increasing its fluorescence, the structural context of the chromophore in GFP can alter its properties in other ways. For example, by mutating amino acids within and near the chromophore in the tertiary structure of the protein, it has been possible to generate blue, cyan, and yellow versions of GFP as well as variants with shifted absorption spectra.3,8 These variants have been particularly useful for FRET studies in which a GFP with a given maximum emission wavelength is tethered to a second GFP with an overlapping excitation wavelength by a linker that changes its conformation in the presence of a ligand of interest.3 GFP is widely used as a genetic reporter to analyze protein expression and localization.9 It is also a powerful tool in bioimaging applications such as fluorescence microscopy.9 The importance of GFP in biotechnology and basic research has stimulated the search for GFP-like nucleic acid structures that generate fluorescence. The first example of such a motif was discovered during the characterization of aptamers that © 2018 American Chemical Society

Received: March 1, 2018 Revised: June 8, 2018 Published: June 13, 2018 4052

DOI: 10.1021/acs.biochem.8b00252 Biochemistry 2018, 57, 4052−4062

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Figure 1. Effect of mutations in tetrads on G-quadruplex fluorescence. (A) Primary sequence and proposed topology of the reference construct used in these experiments. Mutated positions in the central tetrad and loops are numbered. (B) Fluorescence spectrum of the reference construct used in these experiments (blue curve, reference) compared to a random sequence pool of the same length (orange curve, random). (C) Maximum fluorescence intensity of the reference construct compared to that of a 17-nucleotide random sequence pool. (D) Heat map showing the relative fluorescence intensity of all possible variants of the central tetrad in the reference G-quadruplex. (E) Maximum emission wavelength of all possible variants of the central tetrad in the reference G-quadruplex. Experiments were performed at a 10 μM G-quadruplex concentration in a buffer containing 1 M KCl and 20 mM HEPES (pH 7.1) using an excitation wavelength of 290 nm. Experiments in panels B and C were performed using a G-quadruplex with the sequence GGGTGGGAAGGGTGGGA. See Table S1 for more information about the sequences, fluorescence intensities, and maximum emission wavelengths of these constructs.

quadruplexes. Guanine is weakly fluorescent by itself,22 and this is likely enhanced by the extended system of conjugation generated by a tetrad.23−25 Stabilization of tetrads by stacking interactions probably also plays a role by restricting molecular motions that inhibit fluorescence.26 The relative orientations of tetrads are also thought to be important25,27 and probably explain why multimerization modulates the fluorescence of some G-quadruplexes.28−30 Fluorescence anisotrophy experi-

ments suggest that energy transfer occurs among the bases in fluorescent G-quadruplexes, and time-resolved studies indicate that the fluorescence lifetimes of G-quadruplexes are longer than those of nucleotides.25,31,32 Although the signal generated by fluorescent G-quadruplexes is only ∼20-fold above background, with a fluorescence quantum yield of approximately 10−3,19,33 their sequence requirements have been explored to only a limited extent.24 This raises the possibility 4053


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these examples, fluorescence intensities (measured as described above) reflected the fluorescence quantum yields. Native Gels. In a typical assay, material from a 100 μM Gquadruplex stock solution was mixed with a trace amount (≤10 nM) of a radiolabeled version of the sequence. The solution was then heated at 65 °C for 5 min, cooled at room temperature for 5 min, and mixed with buffer. Final concentrations were 10 μM G-quadruplex in a buffer containing 1 M KCl and 20 mM HEPES (pH 7.1). After incubating at room temperature for 30 min, the material was analyzed on 10% native PAGE gels containing 5 mM KCl in both the gel and the buffer. Gels were run at 300 V for 30 min and scanned using a Typhoon phosphorimager. For more information, see refs 34 and 35.

that more comprehensive searches of sequence space could provide new insights into the mechanism by which fluorescence is enhanced and also identify variants with improved fluorescent properties. To investigate these possibilities, we measured the fluorescence intensity and maximum emission wavelengths of approximately 500 variants of a reference G-quadruplex structure. Our library contained mutations in tetrads, loops, and overhanging nucleotides, and most mutations were present in several sequence backgrounds. A handful of mutants in this library were slightly more fluorescent than the reference G-quadruplex, and others had maximum emission wavelengths that were shifted relative to the starting construct. Mutations that increased the fluorescence intensity were distinct from those that increased the maximum emission wavelength, suggesting a trade-off between these two biochemical properties. The fluorescence intensity and maximum emission wavelength were also correlated with the multimeric state: the most fluorescent G-quadruplexes were monomers, while those with the highest maximum emission wavelengths typically formed dimeric structures. We also explored several strategies for enhancing the fluorescence intensity of G-quadruplex structures. These experiments revealed that in some cases concatemerization can modestly increase the fluorescence of oligonucleotides containing multiple G-quadruplexes, although this enhancement depends on both the length and sequence of the spacer linking the Gquadruplexes. Taken together, our results provide new insights into the properties of fluorescent G-quadruplexes and should aid in the development of improved label-free nucleic acid sensors.

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RESULTS AND DISCUSSION Fluorescence of G-Quadruplexes with Mutated Tetrads. In several recent studies, we investigated the effects of mutating the central tetrad in a parallel-strand G-quadruplex on its ability to bind GTP, promote peroxidase reactions, and form multimeric structures.34−36 The reference construct used in these experiments is fluorescent (Figure 1A−C and Figure S3), and we speculated that mutations in tetrads could affect its spectroscopic properties. We were motivated in part by the idea that the fluorescence of G-quadruplexes is enhanced by the extended conjugation of GGGG tetrads.23−25 If true, Gquadruplexes containing mutations in tetrads, especially those that form noncanonical tetrads, might exhibit unusual fluorescent properties. Moreover, because stacking of tetrads at the interfaces of multimeric G-quadruplexes can in some cases alter their properties,28−30 we hypothesized that mutations in our library previously shown to induce formation of higher-order structures35 might also affect G-quadruplex fluorescence. To address these questions in a systematic way, we characterized the fluorescence of all possible sequence variants of the central tetrad of our reference G-quadruplex. Each of these 256 variants was folded in a buffer optimized with respect to DNA concentration, potassium concentration, and pH (Figure S4). After excitation at a wavelength of 290 nm (the optimal excitation wavelength of the reference construct), emission was measured between 330 and 500 nm. This revealed that, as is the case for G-quadruplexes with other biochemical activities,34,35 approximately 10% of these mutants were above our cutoff for fluorescence intensity, including several examples as fluorescent as the reference construct (Figure 1D). The sequence requirements of fluorescent G-quadruplexes were more similar to those of Gquadruplexes that bind GTP and form tetramers than to those that promote peroxidase reactions and form dimers. In particular, all G-quadruplexes containing a GGNN mutation in the central tetrad were above our cutoff for fluorescence intensity (compare to Figure 3 of ref 34 and Figure 5 of ref 35). We also noticed that the emission spectra of some variants were shifted relative to that of the reference construct (Figure 1E and Figure S5). This was most pronounced for three variants with emission peaks >20 nm higher than that of the reference construct (Figure 1E and Figure S5). In contrast to the most fluorescent mutants in the library, each of these variants contained an NNGG rather than a GGNN mutation in the central tetrad of the reference construct (Figure 1E). Control experiments indicated that the excitation wavelengths of these variants were not also shifted (Figure S6). Taken together, these experiments indicate that mutations in the

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MATERIALS AND EXPERIMENTAL DETAILS Reagents. Desalted DNA oligonucleotides, salts, and buffer were purchased from Sigma. Oligonucleotides were resuspended in Milli-Q water at a concentration of 100 μM and typically used without additional purification. Control experiments indicated that the fluorescent properties of these oligonucleotides were similar before and after high-performance liquid chromatography purification (Figure S1). Longer oligonucleotides (i.e., those used in concatamerization experiments) were purified on polyacrylamide gel electrophoresis (PAGE) gels. Stock solutions were stored at −20 °C and thawed at room temperature before use. Fluorescence Measurements. In a typical assay, a 100 μM G-quadruplex stock solution (stored at −20 °C) was thawed at room temperature. After the solution was vortexed, 15 μL was mixed with 60 μL of Milli-Q water. The solution was then heated at 65 °C for 5 min, cooled at room temperature for 5 min, and mixed with 75 μL of 2× buffer. Final concentrations were 10 μM G-quadruplex in a buffer containing 1 M KCl and 20 mM HEPES (pH 7.1). After incubating for 30 min, the sample was excited at 290 nm, and the emission spectrum was typically measured from 330 to 500 nm using a FluoroMax-4 spectrofluorometer (Horiba Scientific). Some of the measurements in Figure 1D were made using a Spark fluorescent plate reader (Tecan), and we confirmed that results were similar to those obtained using the spectrofluorometer (Figure S2). The background fluorescence was determined by measuring the emission spectrum of a sample containing buffer alone and was subtracted from each G-quadruplex measurement. Fluorescence quantum yields were also measured for several constructs (Figure S3). For 4054


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Figure 2. Effect of mutations in loops on G-quadruplex fluorescence intensity. (A) Effects of point mutations in loop positions 4, 8, 9, and 13 on the fluorescence intensity of the reference G-quadruplex. Mutations were made in the context of the sequence GGGTGGGAAGGGTGGGA. (B) Heat map showing the effects of all possible mutations (A, C, or T but not G) in loop positions 4, 8, 9, and 13 on the fluorescence intensity of the reference G-quadruplex described in panel A. (C and D) Same as panels A and B, respectively, but mutations were made in the context of a dimeric G-quadruplex with the sequence GAGTGGGAAGGGTGGGA. (E and F) Same as panels A and B, respectively, but mutations were made in the context of a tetrameric G-quadruplex with the sequence GGGTGGGAAGAGTGGGA. Experiments were performed at a 10 μM G-quadruplex concentration in a buffer containing 1 M KCl and 20 mM HEPES (pH 7.1) using an excitation wavelength of 290 nm. The heights of bars in panels A, C, and E indicate the average of three experiments, and error bars indicate one standard deviation. See Table S1 for more information about the sequences, fluorescence intensities, and maximum emission wavelengths of these constructs. 4055


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Figure 3. Effect of mutations in loops on G-quadruplex maximum emission wavelength. (A) Effects of point mutations in loop positions 4, 8, 9, and 13 on the maximum emission wavelength of the reference G-quadruplex. Mutations were made in the context of the sequence GGGTGGGAAGGGTGGGA. (B) Heat map showing the effects of all possible mutations (A, C, or T but not G) in loop positions 4, 8, 9, and 13 on the maximum emission wavelength of the reference G-quadruplex described in panel A. (C and D) Same as panels A and B, respectively, but mutations were made in the context of a dimeric G-quadruplex with the sequence GAGTGGGAAGGGTGGGA. (E and F) Same as panels A and B, respectively, but mutations were made in the context of a tetrameric G-quadruplex with the sequence GGGTGGGAAGAGTGGGA. Experiments were performed at a 10 μM G-quadruplex concentration in a buffer containing 1 M KCl and 20 mM HEPES (pH 7.1) using an excitation wavelength of 290 nm. The heights of bars in panels A, C, and E indicate the average of three experiments, and error bars indicate one standard deviation. See Table S1 for more information about the sequences, fluorescence intensities, and maximum emission wavelengths of these constructs. 4056


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Figure 4. Effect of overhanging nucleotides on G-quadruplex fluorescence. (A) Proposed secondary structure of a monomeric reference Gquadruplex with the sequence GGGTGGGAAGGGTGGG. (B) Effect of overhanging nucleotides on the fluorescence intensity of this Gquadruplex. (C) Effect of overhanging nucleotides on the maximum emission wavelength of this G-quadruplex. (D) Proposed secondary structure of a dimeric reference G-quadruplex with the sequence GAGTGGGAAGGGTGGG. Note the potentially unstable isolated 5′ tetrad. (E) Effect of overhanging nucleotides on the fluorescence intensity of this G-quadruplex. (F) Effect of overhanging nucleotides on the maximum emission wavelength of this G-quadruplex. (G) Proposed secondary structure of a tetrameric reference G-quadruplex with the sequence GGGTGGGAAGAGTGGG. Note the potentially unstable isolated 3′ tetrad. (H) Effect of overhanging nucleotides on the fluorescence intensity of this G-quadruplex. (I) Effect of overhanging nucleotides on the maximum emission wavelength of this G-quadruplex. Experiments were performed at a 10 μM G-quadruplex concentration in a buffer containing 1 M KCl and 20 mM HEPES (pH 7.1) using an excitation wavelength of 290 nm. The heights of bars indicate the average of three experiments, and error bars indicate one standard deviation. See Table S1 for more information about the sequences, fluorescence intensities, and maximum emission wavelengths of these constructs.

mutations (A, C, or T but not G) at loop positions 4, 8, 9, and 13 in the reference G-quadruplex (Figure 1A). The reference construct also contains a 3′ adenosine overhang (Figure 1A), but this position was not mutated in our library. Because G-quadruplex fluorescence can be affected by multimerization,28−30 mutations were analyzed in two additional sequence backgrounds: that of a dimer-forming sequence that contains an AGGG mutation in the central tetrad of the reference construct and that of a tetramer-forming sequence that contains a GGAG mutation in the central tetrad of the reference construct. As was previously observed for Gquadruplexes that bind GTP, promote peroxidase reactions, and form multimeric structures,34,35 point mutations in loops typically had only small effects on the fluorescence intensity of these G-quadruplexes (Figure 2). However, when G-quadruplexes contained multiple mutations in loops, results were

central tetrad of the reference construct can alter both its fluorescence intensity and its maximum emission wavelength. They also suggest that mutations that enhance fluorescence are distinct from those that modulate the maximum emission wavelength. Fluorescence of G-Quadruplexes with Mutated Loops. We next turned our attention to the effects of mutations in loops on G-quadruplex fluorescence. A previous study of G-quadruplexes with poly(A), poly(C), and poly(T) loops of different lengths showed that G-quadruplexes with shorter loops tend to be more fluorescent than those with longer ones and that that G-quadruplexes with adenosine-rich loops tend to have maximum emission wavelengths higher than those with other sequences.24 To investigate the effects of loop sequences on G-quadruplex fluorescence in a more systematic way, a library was prepared that contained all possible 4057


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were added to model constructs with different multimeric states.34,35 As was the case for mutations in loops, the effects of overhanging nucleotides depended on both the sequence background and the biochemical property being tested (Figure 4). Such overhangs modestly increased the fluorescence of the monomeric reference construct but had no effect on its maximum emission wavelength (Figure 4A−C). In contrast, the addition of overhanging nucleotides to the 3′ (but not 5′) terminus of a dimeric G-quadruplex slightly decreased its fluorescence while significantly increasing its maximum emission wavelength (Figure 4D−F). This was most pronounced for a variant containing a 3′ adenosine, which had an emission peak 50 nm higher than that of a G-quadruplex lacking this adenosine (Figure 4F). The overhang does not change the multimeric state of the Gquadruplex,35 indicating that this is not the reason for the increase in the maximum emission wavelength. The shift in maximum emission wavelength was also observed for a heterodimer made up of one strand with a 3′ adenosine overhang and one strand without it, indicating that a single adenosine was sufficient for this effect (Figure S8). Consistent with this interpretation, dimers containing two or three adenosines at each 3′ terminus did not have maximum emission wavelengths higher than that of a construct containing a single adenosine (Figure S10). Overhanging nucleotides also increased maximum emission wavelengths in the context of a tetrameric G-quadruplex, but effects were smaller and appeared to depend more on the identity of the added nucleotide than on the position of the overhang (Figure 4G−I). In each background, mutational effects of 5′ and 3′ overhangs were independent (Figure S9). This is consistent with previously proposed models, which suggest that 5′ and 3′ termini are on opposite ends of both monomeric and multimeric structures (ref 35 and Figure 4). The patterns observed here were different in many cases from those reported in a previous study, in which 5′ overhangs decreased maximum emission wavelengths while 3′ overhangs either increased maximum emission wavelengths or had no effect.33 Taken together, these results indicate that overhanging nucleotides can modulate the fluorescence properties of Gquadruplexes in complex ways. They also highlight the important role played by the sequence background in determining their effects on G-quadruplex fluorescence. Trade-off between Fluorescence Intensity and Maximum Emission Wavelength. The sequence requirements of the most fluorescent G-quadruplexes identified in this study were distinct from those with the highest maximum emission wavelengths, suggesting a trade-off between these two properties. Some of these differences could be rationalized by sequence background, but loop sequences were also important (Figure 5A,B and Figure S11). For example, the most fluorescent G-quadruplexes in the loop library usually contained a central GGGG tetrad, a C or T at position 4, and a T at position 8 (Figure 5B and Figure S11). On the other hand, G-quadruplexes with high maximum emission wavelengths typically contained an AGGG mutation in the central tetrad of the reference construct and an A at position 4 (Figure 5B and Figure S11). The fluorescence intensity and maximum emission wavelength were also correlated with different multimeric states: the most fluorescent G-quadruplexes in the library were monomers (Figure 5C), while those with the highest maximum emission wavelengths typically formed dimeric structures (Figure 5D). In addition, the range of

typically different in the three sequence backgrounds (Figure 2 and Figure S7). The range of mutational effects was smallest in the context of the reference construct: all variants containing multiple mutations in loops were above our cutoff for fluorescence intensity, and the signal generated by the least fluorescent variant was approximately half of that of the most fluorescent variant (Figure 2B). On the other hand, fluorescence intensities varied >5-fold in both the dimerforming and tetramer-forming backgrounds. Furthermore, ∼10% of variants containing multiple mutations in loops were below our cutoff for fluorescence intensity in the background of a dimer-forming sequence (Figure 2D), and ∼70% were below our cutoff in the background of a tetramerforming sequence (Figure 2F). These differences suggest that loop nucleotides play more important roles in multimers than in monomers. This could be related to our previous observation that these multimers are less stable than the multimeric reference construct35 and might also reflect the number of mutated positions in each type of structure (a DNA strand containing a point mutation will be present in four copies in a tetramer and two copies in a dimer, but in only one copy in a monomer). Effects of mutations in loops on maximum emission wavelengths were also typically background-specific and largest in the dimeric sequence background (Figure 3). Sixteen mutations increased the maximum emission wavelength of the reference G-quadruplex by >10 nm in this background, and seven mutations increased it by >20 nm (Figure 3D). In contrast, only four loop mutations had this effect in the background of the monomeric reference construct (Figure 3B), and none did in the tetrameric sequence background (Figure 3F). A heterodimer made up of constructs with different maximum emission wavelengths had a maximum emission wavelength equal to the average of that of the two homodimers, suggesting that these changes behave like partially dominant mutations (Figure S8). When G-quadruplexes contained multiple mutations in loops, both the fluorescence intensity and the maximum emission wavelength could be approximated using a model in which mutational effects at different loop positions are independent (Figure S9; see also refs 34, 37, and 38). This is consistent with the idea that the nucleotides in the short loops of these G-quadruplexes do not physically interact with one another, although this conclusion cannot necessarily be applied to G-quadruplexes with longer loops. Taken together, these experiments indicate that loop nucleotides can affect the fluorescent properties of Gquadruplexes. They also show that this is dependent on both the sequence background and the biochemical property being tested. Effect of Overhanging Nucleotides on G-Quadruplex Fluorescence. To gain additional insight into the sequence requirements of fluorescent G-quadruplexes, we investigated the extent to which overhanging nucleotides could affect their properties. Such overhangs can inhibit multimerization by interfering with stacking interactions,35,39−43 which in some cases can modulate G-quadruplex fluorescence.28−30 Nonguanosine nucleotides in G-quadruplexes can also interact with tetrads to form unusual structures, including pentads,44 hexads,45 and heptads,46 and such a structure formed by three loop adenosines and a tetrad has been proposed to be responsible for the shifted maximum emission wavelength of the GGAGGAGGAGG G-quadruplex.24 To determine the effects of overhanging nucleotides on the G-quadruplexes studied here, different combinations of 5′ and 3′ overhangs 4058


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in a buffer containing 1 M KCl and 20 mM HEPES (pH 7.1) using an excitation wavelength of 290 nm. See Table S1 for more information about the sequences, fluorescence intensities, and maximum emission wavelengths of these constructs. ss indicates a single-stranded oligonucleotide with the sequence GACTGCCTCGTCACGAT; ds indicates this sequence and its reverse complement.

maximum emission wavelengths in the dimeric sequence background is considerably larger than that observed in the monomeric or tetrameric backgrounds (Figure 5A). The observation that monomers are more fluorescent than multimers could be related to the number of stable tetrads per molecule in each type of structure. Previous work suggests that these monomers contain three stable tetrads per molecule, while dimers and tetramers contain fewer because in each case, at least one of the terminal tetrads is unstable.35 If the fluorescence intensity of a G-quadruplex is related to the number of stable tetrads it contains, monomers would be expected to be more fluorescent than dimers or tetramers, as was observed in this study. The extent of folding could also influence fluorescence but appears to be less important than multimeric state for the G-quadruplexes analyzed here. For example, fluorescence intensity is only weakly correlated with the height of the ∼260 nm peak in the circular dichroism spectra of these G-quadruplexes (Figure S12).47 Although the height of this peak is not a direct readout of the fraction of the folded G-quadruplex (it also reflects the multimeric state of the G-quadruplex and the number of tetrads per folded structure),48 similar results were obtained when only Gquadruplexes with a given multimeric state were analyzed (Figure S12). The observation that dimers tend to have maximum emission wavelengths higher than those of monomers or teramers is more difficult to rationalize because the mechanisms by which the maximum emission wavelength of a G-quadruplex is determined are not well understood.33 Several recent studies suggest that excimers generated by stacking interactions can modulate the fluorescent properties of G-quadruplexes,28−30 and interactions between tetrads and nucleotides in loops have also been proposed to be responsible for shifted maximum emission wavelengths in some Gquadruplexes.24 Although our results do not clearly distinguish between these two possibilities, they do demonstrate that significant changes in the maximum emission wavelength can be achieved in the absence of changes in the multimeric state (Figures 4F and 9A of ref 35). Furthermore, because such shifts occur only when an overhanging nucleotide is close to a stable tetrad (i.e., 3′ overhangs increase the maximum emission wavelengths of dimers, while 5′ overhangs have no effect), it is most consistent with the idea that interactions between unpaired nucleotides and tetrads can shift the maximum emission wavelength of fluorescent G-quadruplexes.24 Effect of Concatamerization on G-Quadruplex Fluorescence. After characterizing the effects of mutations in tetrads, loops, and overhanging nucleotides on the properties of fluorescent G-quadruplexes, we next investigated ways to increase the fluorescence intensity of these structures. The biochemical activities of functional nucleic acids can sometimes be increased by generating constructs that contain multiple copies of a motif. For example, the GTP-binding activity of RNA oligonucleotides containing the CA motif GTP aptamer increases with repeat number.49 This strategy has also

Figure 5. Mutations that increase G-quadruplex fluorescence intensity and maximum emission wavelength. (A) Relationship between maximum emission wavelength and fluorescence intensity for Gquadruplexes with mutations in loops. Blue denotes variants with a central GGGG tetrad and HHHH loop sequences (H = A, C, or T). Orange denotes variants with an AGGG mutation in the central tetrad of the reference G-quadruplex and HHHH loop sequences. Green denotes variants with an GGAG mutation in the central tetrad of the reference G-quadruplex and HHHH loop sequences. (B) The sequence requirements of the most fluorescent G-quadruplexes in the loop library are distinct from those with the highest maximum emission wavelengths. Blue denotes variants with a GGGG central tetrad and YTHH loop sequences (Y = C or T, and H = A, C, or T). Orange denotes variants with an AGGG mutation in the central tetrad of the reference G-quadruplex and AHHH loop sequences. (C) Multimeric state of the 10 most fluorescent G-quadruplex variants in the loop library as determined by native PAGE. (D) Multimeric state of the 10 G-quadruplex variants in the loop library with the highest maximum emission wavelengths as determined by native PAGE. Experiments were performed at a 10 μM G-quadruplex concentration 4059


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Figure 6. Effect of concatemerization on G-quadruplex fluorescence intensity. (A) Design of constructs containing multiple copies of a fluorescent G-quadruplex linked by spacers that vary in both length and sequence. (B) Effect of spacer length and sequence on the fluorescence intensity of oligonucleotides containing two copies of a reference G-quadruplex with the sequence GGGTGGGAAGGGTGGG. (C) Effect of spacer length and sequence on the fluorescence intensity of oligonucleotides containing three copies of a reference G-quadruplex with the sequence GGGTGGGAAGGGTGGG. (D) Fluorescence spectra of oligonucleotides containing two copies of the reference G-quadruplex linked by poly(A) spacers of different lengths. (E) Effect of copy number on the fluorescence intensity of concatemerized G-quadruplexes connected by 20 nucleotide spacers. The bars labeled theoretical maximum indicate the expected values for a model in which each G-quadruplex makes an independent contribution to the fluorescence of the oligonucleotide. Experiments were performed at a 10 μM G-quadruplex concentration in a buffer containing 1 M KCl and 20 mM HEPES (pH 7.1) using an excitation wavelength of 290 nm. See Table S1 for more information about the sequences, fluorescence intensities, and maximum emission wavelengths of these constructs.

sequence and length influence the fluorescence intensity of concatamerized G-quadruplexes. Concatamers containing adenosine linkers were slightly more fluorescent than those containing thymidine linkers and significantly more fluorescent than those containing cytosine linkers (Figure 6B,C). For constructs with adenosine or thymidine linkers, the fluorescence intensity increased with increasing linker length between one and ten nucleotides but typically did not increase for constructs with longer linkers (Figure 6B−D). On the other hand, the fluorescence intensity decreased with linker length for constructs containing cytosine loops, probably because of base pairing between cytosines and guanosines that form tetrads in G-quadruplexes (Figure 6B,C). In the case of constructs that contained adenosine or thymidine linkers of 10

been reported to increase the fluorescent signal generated by the malachite green aptamer.50 On the other hand, the addition of a flanking sequence to an aptamer or ribozyme can be inhibitory, presumably because it increases the propensity of the motif to misfold.51 Furthermore, stacking can quench the signal generated by fluorescent nucleotide analogues such as 2aminopurine,52 and it could have a similar effect on arrays of stacked G-quadruplexes. For these reasons, the expected effect of concatamerization on G-quadruplex fluorescence intensity was not obvious. To address this question experimentally, we measured the fluorescence intensity of a series of constructs in which either two or three copies of the reference G-quadruplex were connected by linkers that differ in terms of both sequence and length (Figure 6A). This revealed that both linker 4060


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or 20 nucleotides, addition of a second G-quadruplex resulted in a 2-fold increase in the intensity of the fluorescent signal, but increases were more modest when a third G-quadruplex was added to the construct (Figure 6B−E). Synthesis of longer constructs could not be readily achieved using either chemical or enzymatic methods. This was likely partially due to the inhibitory effects of G-quadruplexes on DNA synthesis, which can lead to inefficient polymerization as well as amplification bias in the context of methods such as the polymerase chain reaction.53,54 Although encouraging in some respects, these findings also emphasize the need for significant improvements before fluorescent G-quadruplexes can be used as sensors. In particular, the fluorescence quantum yields of the most fluorescent G-quadruplexes identified in this study (Figure S3) are still several orders of magnitude lower than that of GFP,19,33,55 and the optimization of this parameter will represent a significant challenge for future research.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.8b00252. Figures S1−S12 and Table S1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Edward A. Curtis: 0000-0003-2680-0770 Author Contributions

T.M., T.S., and E.A.C. designed the experiments. T.M., T.S., and L.B. performed the experiments. E.A.C. wrote the manuscript. All authors have given approval to the final version of the manuscript. Funding

This work was supported by an IOCB start-up grant awarded to E.A.C., InterBioMed LO 1302 from the Ministry of Education of the Czech Republic, and “Chemical biology for drugging undruggable targets (ChemBioDrug)” (CZ.02.1.01/ 0.0/0.0/16_019/0000729) from the European Regional Development Fund (OP RDE). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Sofia Kolesnikova for useful discussions and assistance with native PAGE and Jan Konvalinka and colleagues at the IOCB for useful discussions and support.

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ABBREVIATIONS RNA, ribonucleic acid; GFP, green fluorescent protein; HBI, 4(p-hydroxybenzylidene)imidazolidin-5-one; FRET, Fö rster resonance energy transfer; SAM, S-adenosylmethionine; ADP, adenosine diphosphate; GTP, guanosine triphosphate.

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REFERENCES

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Sequence Requirements of Intrinsically Fluorescent G

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