Chapter 8 A Fluorescence Correlation Spectroscopy-Based Enzyme Assay for Human Dicer Eileen Magbanua and Ulrich Hahn Abstract We used fluorescence correlation spectroscopy (FCS) to establish an in vitro assay to investigate RNase activity of human Dicer (Werner et al., Biol Chem 393(3):187–193). FCS allows investigating the interactions of different particles due to their differing diffusion mobility, provided that one of the interacting partners exhibit a fluorescence label. In our case we used a fluorophore-labeled double-stranded RNA (dsRNA) as substrate to monitor Dicer activity. The dsRNA was cleaved by the enzyme resulting in a fivenucleotide-short single-stranded RNA (ssRNA) fragment carrying the fluorophore, which could be distinguished from the substrate and unlabeled second product by FCS. Furthermore, we refer to additional (control) experiments to confirm obtained data. Key words FCS, Human Dicer, Dicer assay, RNase III, dsRNA
1 Introduction Fluorescence correlation spectroscopy (FCS) is a method, which enables analyzing fluctuations of diffusing fluorescent molecules. Different fluorescent molecules which vary in mass by a factor of eight can be distinguished from each other [1]. Fulfilling this requirement FCS can easily be used to investigate interactions of fluorescently labeled particles and for example in vitro enzyme activity assays [2–4]. Further advantages of FCS are the small sample amounts as well as a small detection volume of approximately 0.1 fl, which represents less than the volume of a standard bacterial cell. Here we used FCS to investigate the RNase activity of human Dicer [5]. Dicer plays a key role during gene regulation by RNA interference (RNAi). It is responsible for the delivery of substrate for RISC by cleaving double-stranded RNA (dsRNA) to small interfering RNAs (siRNA) or microRNAs (miRNAs) which are than interacting with further proteins to target selected mRNAs resulting in gene silencing [6–8].
Christoph Arenz (ed.), miRNA Maturation: Methods and Protocols, Methods in Molecular Biology, vol. 1095, DOI 10.1007/978-1-62703-703-7_8, © Springer Science+Business Media New York 2014
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Eileen Magbanua and Ulrich Hahn
In this assay we used a part of the coding region of glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) as Dicer substrate. The substrate was built up of an unlabeled sense strand, which was hybridized to an antisense strand fluorescently labeled at its 5′ end (Fig. 1a). To facilitate optimal substrate binding as well as Dicer cleavage we designed a dsRNA containing a typical two- nucleotide 3′ overhang [9, 10]. The substrate was cleaved into three smaller products: a non-fluorescent 21-nucleotide dsRNA, a uridine 5′ phosphate, and a fluorescing five-nucleotide-short single-stranded RNA (ssRNA) (Fig. 1b). The plotting of resulting autocorrelation curves of substrate and cleavage product demonstrates differences in diffusion time (Fig. 2). In addition to the assay outlined several control experiments can be performed. The usage of two different fluorophores verifies
Fig. 1 Dicer substrate. (a) dsRNA Dicer substrate deduced from the coding region of GAPDH mRNA with a two-nucleotide protruding 3′ end. Antisense RNA strand possesses a fluorescence label at its 5′-end; sense strand is not labeled. Arrows indicate putative cleavage sites. (b) Cleavage products after incubation with Dicer are a non-fluorescent 21-nucleotide dsRNA, a uridine 5′ phosphate, and a fluorescing five-nucleotide-long ssRNA
Fig. 2 Autocorrelation functions. Experimentally determined autocorrelation functions of Dicer substrate labeled with ATTO647N (open boxes) and cleavage product (filled boxes). Arrow indicates that components with lower molecular weight (MW) show autocorrelation function curves shifted to the left
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Dicer activity and reduces the risk of artifacts due to unspecific interactions or photodynamic processes. As Dicer activity is strongly dependent on the presence of Mg2+-ions for instance replacement of Mg2+- by Ca2+-ions leads to a significant inhibition of Dicer activity [11], and thus diffusion time should remain constant due to remaining uncleaved substrate even after enzyme addition. Furthermore, Dicer does solely cleave dsRNA [9]. Therefore fluorescently labeled ssRNA can be used as control as well. Bacterial RNase III generates Dicer-like termini and overhangs after cleavage of dsRNA, but cleavage products differ in size. RNase III of Escherichia coli cleaves dsRNA into 12–15-nucleotide-long RNA fragments. Using GAPDH-derived dsRNA substrate (Fig. 1a) RNase III produces RNA fragments that differ in size from Dicer products. Significant differences should be observed comparing autocorrelation curves as well as diffusion coefficients of cleavage products derived of both enzymes.
2 Materials Prepare all solutions RNase free (see Note 1). 2.1 Cleavage Assay
1. Recombinant human Dicer (Genlantis). 2. E. coli RNase III (e.g., New England Biolabs). 3. Dicer substrates: Sense RNA (5′-AAGGCUGAGAACGGGAAGCUUU-3′). Antisense RNA 5′-ATTO647N (5′-ATTO647N-AGUUCC GACUCUUGCCCUUCGAAAAG-3′) (see Note 2). Antisense RNA 5′-Cy5 (5′-Cy5-AGUUCCGACUCUUGCC CUUCGAAAAG-3′). 4. Buffer A: 30 mM Tris–HCl, pH 6.8, 50 mM NaCl. 5. Buffer B: 20 mM Tris–HCl, pH 8.0, 250 mM NaCl. 6. Buffer C: 10 mM Tris–HCl, pH 7.5, 350 mM KCl, 0.1 mM EDTA, 0.1 mM DTT, 2.5 mM MgCl2. 7. 100 mM MgCl2 stock solution. 8. 100 mM CaCl2 stock solution. 9. RNase-free water.
2.2 FCS Measurement
1. Confocal laser scanning microscope (cLSM) with photon counting unit (e.g., ConforCor2 (Carl Zeiss) with software package) (see Note 3). 2. Cover slip 24 × 50 mm (CarlRoth) (see Note 4).
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Eileen Magbanua and Ulrich Hahn
3 Methods 3.1 RNA Denaturation and Renaturation
1. Dilute ATTO647N-labeled RNA in Buffer A and combine it with sense RNA equimolecular. 2. Dilute Cy5-labeled RNA in Buffer B and combine it with sense RNA equimolecular. 3. Heat RNA for 3 min at 95 °C. 4. Cool sample down by 1 °C steps per 1.2 min until a temperature of 25 °C is reached. 5. Add MgCl2 to a final concentration of 2.5 mM (see Note 5).
3.2 FCS Configuration for ATTO647N and Cy5 (See Note 6)
1. Excite with 3 % of 5.0 mW of a HeNe laser at 633 nm. 2. Use the water immersion objective C-Apochromat 40× with numerical aperture of 1.2. 3. Separate emitted light from detection volume with a 633 nm main dichromic mirror (HFT) and 650 nm emission long-pass filter. 4. Adjust pinhole to 90 μm. 5. Measurement period: 5 or 10 s.
3.3 FCS Measurements
Measurements were carried out in a sample volume of 20 μl positioned on cover slide in cLSM at room temperature. 1. Measure diffusion time of 5 nM fluorescent dsRNA in the absence of enzyme in appropriate buffer. 2. Add Dicer to a final concentration of 1 U directly to the substrate on cover slide, and measure diffusion time again. Diffusion time decreases significantly due to generation of cleavage product (see Note 7). 3. Plot autocorrelation curves of substrate and Dicer product against diffusing time and compare (Fig. 2). The smaller the molecular weight of a diffusing component the more the autocorrelation curve is shifted to the left. 4. Optional control experiment 1: Perform Dicer assay with another Dicer substrate, which is labeled with an alternative fluorophore. 5. Optional control experiment 2: After RNA denaturation and renaturation add CaCl2 to a final concentration of 2.5 mM instead of MgCl2 to RNA (see Note 8). Then perform Dicer assay as described in steps 1–3. No reduction of diffusion time should be observed. 6. Optional control experiment 3: Perform Dicer assay with single-stranded fluorescent RNA as substrate. No reduction regarding diffusion time should be observed (see Note 9).
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7. Optional control experiment 4: Use Cy5-labeled substrate and perform Dicer assay with Dicer enzyme and RNase III in parallel. In case of RNase III use 2.6 U enzyme and buffer C. Subsequently, compare autocorrelation curves of substrate alone with products after Dicer and RNase III cleavage, respectively. Autocorrelation curve should differ significantly due to unequal cleavage products. Define diffusion time τ of cleavage products by using the two-component model. Therefore use instrument software and insert diffusion time of uncleaved substrate as fixed value. The calculated value represents diffusion time of respective cleavage products. Finally, calculate diffusion coefficients D of cleavage products with
D=
r02 4 ⋅t
and use for the radial distance to the center of the laser beam focus r0,Cy5 = 250 nm and diffusion time τ of cleavage product, respectively. Determined diffusion coefficients should differ significantly.
4 Notes 1. For RNase-free water specific filter installation can be used. Optionally solutions can be treated with diethylpyrocarbonate (DEPC) to inactivate RNases due to acetylation of histidine, lysine, cysteine, and tyrosine residues. Therefore solution is treated with 0.1 % (v/v) DEPC and stirred overnight. Autoclaving afterwards leads to destruction of DEPC. 2. Of course RNA can be labeled with two other fluorophores. Ideally both fluorophores should possess same absorption and emission spectra and be suitable for FCS measurements. 3. Using a software package facilitates the usage of FCS as well as analysis of data sets to apply different component models (e.g., two-component model) to calculate diffusion time of a second diffusion compound for instance. Otherwise all parameters would have to be calculated in the old-fashioned way by hand. 4. Thickness of cover slip has to enable performing confocal measurements. 5. Heating RNA in the presence of MgCl2 might lead to hydrolysis of RNA. 6. Using other fluorophores these parameters have to be adjusted.
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7. Investigating two different components by FCS, both have to differ significantly regarding diffusion time or molar mass, respectively. 8. Replacing divalent cations leads to inhibition of Dicer activity. 9. Dicer exclusively cleaves dsRNA and not ssRNA. Nevertheless, diffusion time can increase slightly after Dicer addition due to Dicer affinity to 5′-terminal modifications of ssRNA [12]. References 1. Weisshart K, Jungel V, Briddon SJ (2004) The LSM 510 META - ConfoCor 2 system: an integrated imaging and spectroscopic platform for single-molecule detection. Curr Pharm Biotechnol 5(2):135–154 2. Kinjo M, Nishimura G, Koyama T et al (1998) Single-molecule analysis of restriction DNA fragments using fluorescence correlation spectroscopy. Anal Biochem 260(2):166–172 3. Ketting RF, Fischer SE, Bernstein E et al (2001) Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev 15(20):2654–2659 4. Kohl T, Haustein E, Schwille P (2005) Determining protease activity in vivo by fluorescence cross-correlation analysis. Biophys J 89(4):2770–2782 5. Werner A, Skakun VV, Ziegelmuller P et al (2012) A fluorescence correlation spectroscopy- based enzyme assay for human Dicer. Biol Chem 393(3):187–193 6. Fire A, Xu S, Montgomery MK et al (1998) Potent and specific genetic interference by
double-stranded RNA in Caenorhabditis elegans. Nature 391(6669):806–811 7. Jinek M, Doudna JA (2009) A three-dimensional view of the molecular machinery of RNA interference. Nature 457(7228):405–412 8. Pillai RS, Bhattacharyya SN, Filipowicz W (2007) Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol 17(3):118–126 9. Zhang H, Kolb FA, Brondani V et al (2002) Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J 21(21):5875–5885 10. Vermeulen A, Behlen L, Reynolds A et al (2005) The contributions of dsRNA structure to Dicer specificity and efficiency. RNA 11(5): 674–682 11. Provost P, Dishart D, Doucet J et al (2002) Ribonuclease activity and RNA binding of recombinant human Dicer. EMBO J 21(21): 5864–5874 12. Kini HK, Walton SP (2007) In vitro binding of single-stranded RNA by human Dicer. FEBS Lett 581(29):5611–5616
1 Introduction Fluorescence correlation spectroscopy (FCS) is a method, which enables analyzing fluctuations of diffusing fluorescent molecules. Different fluorescent molecules which vary in mass by a factor of eight can be distinguished from each other [1]. Fulfilling this requirement FCS can easily be used to investigate interactions of fluorescently labeled particles and for example in vitro enzyme activity assays [2–4]. Further advantages of FCS are the small sample amounts as well as a small detection volume of approximately 0.1 fl, which represents less than the volume of a standard bacterial cell. Here we used FCS to investigate the RNase activity of human Dicer [5]. Dicer plays a key role during gene regulation by RNA interference (RNAi). It is responsible for the delivery of substrate for RISC by cleaving double-stranded RNA (dsRNA) to small interfering RNAs (siRNA) or microRNAs (miRNAs) which are than interacting with further proteins to target selected mRNAs resulting in gene silencing [6–8].
Christoph Arenz (ed.), miRNA Maturation: Methods and Protocols, Methods in Molecular Biology, vol. 1095, DOI 10.1007/978-1-62703-703-7_8, © Springer Science+Business Media New York 2014
103
104
Eileen Magbanua and Ulrich Hahn
In this assay we used a part of the coding region of glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) as Dicer substrate. The substrate was built up of an unlabeled sense strand, which was hybridized to an antisense strand fluorescently labeled at its 5′ end (Fig. 1a). To facilitate optimal substrate binding as well as Dicer cleavage we designed a dsRNA containing a typical two- nucleotide 3′ overhang [9, 10]. The substrate was cleaved into three smaller products: a non-fluorescent 21-nucleotide dsRNA, a uridine 5′ phosphate, and a fluorescing five-nucleotide-short single-stranded RNA (ssRNA) (Fig. 1b). The plotting of resulting autocorrelation curves of substrate and cleavage product demonstrates differences in diffusion time (Fig. 2). In addition to the assay outlined several control experiments can be performed. The usage of two different fluorophores verifies
Fig. 1 Dicer substrate. (a) dsRNA Dicer substrate deduced from the coding region of GAPDH mRNA with a two-nucleotide protruding 3′ end. Antisense RNA strand possesses a fluorescence label at its 5′-end; sense strand is not labeled. Arrows indicate putative cleavage sites. (b) Cleavage products after incubation with Dicer are a non-fluorescent 21-nucleotide dsRNA, a uridine 5′ phosphate, and a fluorescing five-nucleotide-long ssRNA
Fig. 2 Autocorrelation functions. Experimentally determined autocorrelation functions of Dicer substrate labeled with ATTO647N (open boxes) and cleavage product (filled boxes). Arrow indicates that components with lower molecular weight (MW) show autocorrelation function curves shifted to the left
Dicer Activity Assay with FCS
105
Dicer activity and reduces the risk of artifacts due to unspecific interactions or photodynamic processes. As Dicer activity is strongly dependent on the presence of Mg2+-ions for instance replacement of Mg2+- by Ca2+-ions leads to a significant inhibition of Dicer activity [11], and thus diffusion time should remain constant due to remaining uncleaved substrate even after enzyme addition. Furthermore, Dicer does solely cleave dsRNA [9]. Therefore fluorescently labeled ssRNA can be used as control as well. Bacterial RNase III generates Dicer-like termini and overhangs after cleavage of dsRNA, but cleavage products differ in size. RNase III of Escherichia coli cleaves dsRNA into 12–15-nucleotide-long RNA fragments. Using GAPDH-derived dsRNA substrate (Fig. 1a) RNase III produces RNA fragments that differ in size from Dicer products. Significant differences should be observed comparing autocorrelation curves as well as diffusion coefficients of cleavage products derived of both enzymes.
2 Materials Prepare all solutions RNase free (see Note 1). 2.1 Cleavage Assay
1. Recombinant human Dicer (Genlantis). 2. E. coli RNase III (e.g., New England Biolabs). 3. Dicer substrates: Sense RNA (5′-AAGGCUGAGAACGGGAAGCUUU-3′). Antisense RNA 5′-ATTO647N (5′-ATTO647N-AGUUCC GACUCUUGCCCUUCGAAAAG-3′) (see Note 2). Antisense RNA 5′-Cy5 (5′-Cy5-AGUUCCGACUCUUGCC CUUCGAAAAG-3′). 4. Buffer A: 30 mM Tris–HCl, pH 6.8, 50 mM NaCl. 5. Buffer B: 20 mM Tris–HCl, pH 8.0, 250 mM NaCl. 6. Buffer C: 10 mM Tris–HCl, pH 7.5, 350 mM KCl, 0.1 mM EDTA, 0.1 mM DTT, 2.5 mM MgCl2. 7. 100 mM MgCl2 stock solution. 8. 100 mM CaCl2 stock solution. 9. RNase-free water.
2.2 FCS Measurement
1. Confocal laser scanning microscope (cLSM) with photon counting unit (e.g., ConforCor2 (Carl Zeiss) with software package) (see Note 3). 2. Cover slip 24 × 50 mm (CarlRoth) (see Note 4).
106
Eileen Magbanua and Ulrich Hahn
3 Methods 3.1 RNA Denaturation and Renaturation
1. Dilute ATTO647N-labeled RNA in Buffer A and combine it with sense RNA equimolecular. 2. Dilute Cy5-labeled RNA in Buffer B and combine it with sense RNA equimolecular. 3. Heat RNA for 3 min at 95 °C. 4. Cool sample down by 1 °C steps per 1.2 min until a temperature of 25 °C is reached. 5. Add MgCl2 to a final concentration of 2.5 mM (see Note 5).
3.2 FCS Configuration for ATTO647N and Cy5 (See Note 6)
1. Excite with 3 % of 5.0 mW of a HeNe laser at 633 nm. 2. Use the water immersion objective C-Apochromat 40× with numerical aperture of 1.2. 3. Separate emitted light from detection volume with a 633 nm main dichromic mirror (HFT) and 650 nm emission long-pass filter. 4. Adjust pinhole to 90 μm. 5. Measurement period: 5 or 10 s.
3.3 FCS Measurements
Measurements were carried out in a sample volume of 20 μl positioned on cover slide in cLSM at room temperature. 1. Measure diffusion time of 5 nM fluorescent dsRNA in the absence of enzyme in appropriate buffer. 2. Add Dicer to a final concentration of 1 U directly to the substrate on cover slide, and measure diffusion time again. Diffusion time decreases significantly due to generation of cleavage product (see Note 7). 3. Plot autocorrelation curves of substrate and Dicer product against diffusing time and compare (Fig. 2). The smaller the molecular weight of a diffusing component the more the autocorrelation curve is shifted to the left. 4. Optional control experiment 1: Perform Dicer assay with another Dicer substrate, which is labeled with an alternative fluorophore. 5. Optional control experiment 2: After RNA denaturation and renaturation add CaCl2 to a final concentration of 2.5 mM instead of MgCl2 to RNA (see Note 8). Then perform Dicer assay as described in steps 1–3. No reduction of diffusion time should be observed. 6. Optional control experiment 3: Perform Dicer assay with single-stranded fluorescent RNA as substrate. No reduction regarding diffusion time should be observed (see Note 9).
Dicer Activity Assay with FCS
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7. Optional control experiment 4: Use Cy5-labeled substrate and perform Dicer assay with Dicer enzyme and RNase III in parallel. In case of RNase III use 2.6 U enzyme and buffer C. Subsequently, compare autocorrelation curves of substrate alone with products after Dicer and RNase III cleavage, respectively. Autocorrelation curve should differ significantly due to unequal cleavage products. Define diffusion time τ of cleavage products by using the two-component model. Therefore use instrument software and insert diffusion time of uncleaved substrate as fixed value. The calculated value represents diffusion time of respective cleavage products. Finally, calculate diffusion coefficients D of cleavage products with
D=
r02 4 ⋅t
and use for the radial distance to the center of the laser beam focus r0,Cy5 = 250 nm and diffusion time τ of cleavage product, respectively. Determined diffusion coefficients should differ significantly.
4 Notes 1. For RNase-free water specific filter installation can be used. Optionally solutions can be treated with diethylpyrocarbonate (DEPC) to inactivate RNases due to acetylation of histidine, lysine, cysteine, and tyrosine residues. Therefore solution is treated with 0.1 % (v/v) DEPC and stirred overnight. Autoclaving afterwards leads to destruction of DEPC. 2. Of course RNA can be labeled with two other fluorophores. Ideally both fluorophores should possess same absorption and emission spectra and be suitable for FCS measurements. 3. Using a software package facilitates the usage of FCS as well as analysis of data sets to apply different component models (e.g., two-component model) to calculate diffusion time of a second diffusion compound for instance. Otherwise all parameters would have to be calculated in the old-fashioned way by hand. 4. Thickness of cover slip has to enable performing confocal measurements. 5. Heating RNA in the presence of MgCl2 might lead to hydrolysis of RNA. 6. Using other fluorophores these parameters have to be adjusted.
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Eileen Magbanua and Ulrich Hahn
7. Investigating two different components by FCS, both have to differ significantly regarding diffusion time or molar mass, respectively. 8. Replacing divalent cations leads to inhibition of Dicer activity. 9. Dicer exclusively cleaves dsRNA and not ssRNA. Nevertheless, diffusion time can increase slightly after Dicer addition due to Dicer affinity to 5′-terminal modifications of ssRNA [12]. References 1. Weisshart K, Jungel V, Briddon SJ (2004) The LSM 510 META - ConfoCor 2 system: an integrated imaging and spectroscopic platform for single-molecule detection. Curr Pharm Biotechnol 5(2):135–154 2. Kinjo M, Nishimura G, Koyama T et al (1998) Single-molecule analysis of restriction DNA fragments using fluorescence correlation spectroscopy. Anal Biochem 260(2):166–172 3. Ketting RF, Fischer SE, Bernstein E et al (2001) Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev 15(20):2654–2659 4. Kohl T, Haustein E, Schwille P (2005) Determining protease activity in vivo by fluorescence cross-correlation analysis. Biophys J 89(4):2770–2782 5. Werner A, Skakun VV, Ziegelmuller P et al (2012) A fluorescence correlation spectroscopy- based enzyme assay for human Dicer. Biol Chem 393(3):187–193 6. Fire A, Xu S, Montgomery MK et al (1998) Potent and specific genetic interference by
double-stranded RNA in Caenorhabditis elegans. Nature 391(6669):806–811 7. Jinek M, Doudna JA (2009) A three-dimensional view of the molecular machinery of RNA interference. Nature 457(7228):405–412 8. Pillai RS, Bhattacharyya SN, Filipowicz W (2007) Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol 17(3):118–126 9. Zhang H, Kolb FA, Brondani V et al (2002) Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J 21(21):5875–5885 10. Vermeulen A, Behlen L, Reynolds A et al (2005) The contributions of dsRNA structure to Dicer specificity and efficiency. RNA 11(5): 674–682 11. Provost P, Dishart D, Doucet J et al (2002) Ribonuclease activity and RNA binding of recombinant human Dicer. EMBO J 21(21): 5864–5874 12. Kini HK, Walton SP (2007) In vitro binding of single-stranded RNA by human Dicer. FEBS Lett 581(29):5611–5616
