1. Volume 23, Issue 12, Pages 1912-1926 (December 2015)
Inhibition of Aggregation of Mutant Huntingtin by Nucleic Acid Aptamers In Vitro and in a Yeast Model of Huntington's Disease 
Rajeev K Chaudhary, Kinjal A Patel, Milan K Patel, Radha H Joshi, Ipsita Roy 
Molecular Therapy 
Volume 23, Issue 12, Pages (December 2015)
DOI: /mt
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
GST-51Q-htt forms fibrillar aggregates. (a) GST-20Q-htt and GST-51Q-htt (1 mg ml−1 each, 40 mmol/l TrisHCl buffer, pH 8.0 containing 150 mmol/l NaCl) were incubated separately at 4 and 37 °C for 144 hours and analyzed by immunoblotting using GST and polyglutamine antibodies, respectively. (b) GST-51Q-htt (1 mg ml−1, 40 mmol/l TrisHCl buffer, pH 8.0 containing 150 mmol/l NaCl) was incubated at 37 °C for different time intervals. Filter retardation assay using cellulose acetate membrane (0.2 μm) was carried out at different time intervals and the amount of aggregates retained was probed with polyglutamine antibody. Triplicate dots are shown for each time point. (c) Densitometric analysis of the intensity of the dots obtained with polyglutamine-specific or A11-specific (blot image shown in Supplementary Figure S1b) antibody was carried out separately with ImageQuant TL software (GE Healthcare). The intensity of the dot at the last point of analysis (72 hours for polyglutamine antibody and 144 hours for A11 antibody) was assigned an arbitrary value of 100%. Inset shows the expanded view of the aggregation curve for the time period of 0–72 hours. (d) The nature of aggregates formed after incubating GST-51Q-htt (1 mg ml−1, 40 mmol/l TrisHCl buffer, pH 8.0 containing 150 mmol/l NaCl) for 15 hours (exponential phase of fibrillation) and 144 hours (saturation phase) at 37 °C was observed by suspending the aggregates in 40 mmol/l TrisHCl buffer, pH 8.0 containing 150 mmol/l NaCl, with or without 2% SDS and carrying out a filter retardation assay. The cellulose acetate membrane was probed with GST antibody. Triplicate dots are shown. The intensity of the dots indicating the amount of aggregates retained on the membrane in the absence of SDS was assigned an arbitrary value of 100%. Values shown are mean ± SEM of three independent experiments. (e) Transmission electron micrograph of GST-51Q-htt incubated for 144 hours as described in the text. Bar = 200 nm; Magnification = 11,500×. (f) GST-51Q-htt (1 mg ml−1, 40 mmol/l TrisHCl buffer, pH 8.0 containing 150 mmol/l NaCl) was incubated at 37 °C and analyzed after indicated time intervals by Thioflavin T fluorescence spectroscopy. The final concentrations of the protein and the fluorophore were 1.5 and 50 μmol/l, respectively. Samples were excited at 440 nm and the emission intensity was monitored at 484 nm. Values shown are mean ± SEM of three independent experiments.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Inhibition of aggregation of GST-51Q-htt by RNA sequences in vitro. (a) Monoclonal RNA sequences obtained from enriched libraries were preincubated with GST-51Q-htt for 1 hour at 25 °C, followed by incubation at 37 °C for 15 hours. Aggregation was monitored by filter retardation assay using a cellulose acetate membrane which was probed with GST antibody. The intensities of protein samples incubated in the presence of RNA sequences are shown as empty bars. Intensity of the control sample (incubated without RNA sequences, c, filled bar) was assigned a value of 100% in all cases. Data represent mean ± SEM of three independent experiments. *P < 0.05, **P < 0.005, ***P < against corresponding control. (b) Incubation of GST-51Q-htt was carried out as before (Figure 2a) and the extent of aggregation was measured with A11-specific antibody following a dot-blot assay on a nitrocellulose membrane. Intensity of the control sample (incubated without RNA sequences, c, filled bar) was assigned a value of 100% in all cases. Data represent mean ± SEM of three independent experiments. **P < 0.005, ***P < against corresponding control (filled bar, in the absence of aptamer). Comparison of binding affinities of (c) mHtt and (d) mHtt for GST-20Q-htt, GST-51Q-htt, and 103Q-htt by dot blot assay. The fluorescence intensities of the retained Qn-htt-RNA complexes were quantified and fitted into the equation for a sigmoidal curve. The intensity of the dot for GST-51Q-htt-RNA at the highest concentration of the protein has been assigned an arbitrary value of 100% and the intensities of the other dots have been calculated with respect to this value. A representative curve from each measurement is shown. (e) The ability of aptamers to bind to preformed GST-51Q-htt aggregates was determined by incubating the aggregates (formed after incubation of GST-51Q-htt at 37 °C for 60 hours) with labeled RNA sequences at 25 °C for 1 hour and filtering the RNA-protein complex through a cellulose acetate membrane. The fluorescence intensity retained on the membrane was quantified by densitometry. The intensity of the aggregate-RNA complex at the starting point in each case was assigned a value of 100% in all cases.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
Inhibition of aggregation of GST-51Q-htt by a combination of RNA sequences. (a) GST-51Q-htt was incubated with one (empty bars) or two (gray bars) aptamers as indicated, at a ratio of 1:1 (w w−1, protein:aptamer) and the extent of aggregation was monitored as described in the legend to Figure 2a. Values shown are mean ± SEM of three independent experiments. ***P < 0.001, **P < 0.005, *P < 0.05 against control (incubated without RNA sequences, c, filled bar). Displacement assay was carried out to determine the overlap of binding sites of the aptamers on the surface of the polyglutamine stretch. Densitometric analysis of the retained fluorescence intensity of the RNA-protein complex was carried out using ImageQuant TL software (GE Healthcare). Results are shown for increasing amounts of unlabeled (b) , (c) , and (d) aptamers, in the presence of constant amount (70 ng) of fluorescein-labeled aptamers, as indicated. The fluorescence intensity of the RNA-protein complex in the absence of any unlabeled aptamer has been arbitrarily assigned a value of 100% in each case. Measurement of mean particle size of GST-51Q-htt by dynamic light scattering after (e) 15 hours and (f) 60 hours of incubation at 37 °C in the absence and presence of different aptamers. Size represents diameter of particles. (g) The ability of aptamers to reverse aggregation was measured by adding a pair of aptamers after incubation of GST-51Q-htt for 15 and 60 hours (as indicated) at 37 °C separately. GST-51Q-htt-RNA complexes were incubated for a further 5 hours (empty bars) or 11 hours (gray bars). In case of control (filled bar), an equivalent volume of diethylpyrocarbonate water was added instead of RNA sequences. The extent of aggregation was monitored as described in the legend to Figure 2a. Intensity of the control sample (incubated without RNA sequences, c, filled bar) was assigned a value of 100% in all cases. Values shown are mean ± SEM of three independent experiments.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Effect of RNA aptamers on aggregation of GST-51Q-htt. (a) Release of encapsulated fluorescein dye from liposomes was monitored in the presence of monomeric (filled bar), oligomeric (empty bars) or fibrillar (gray bars) GST-51Q-htt incubated in the absence and presence of mHtt (striped bars) and mHtt (dotted bars) for 20 minutes at 37 °C. (b) Monomeric, (c) oligomeric, and (d) fibrillar GST-51Q-htt were incubated with rat RBCs in the absence and presence of mHtt for different time periods at 37 °C and oxidative stress in RBCs was measured using DCFH-DA assay. Values shown are mean ± SEM of three independent experiments. ***P < 0.001, **P < 0.01 against the corresponding monomeric GST-51Q-htt, $$$P < 0.001, $P < 0.05 against oligomeric GST-51Q-htt (in the absence of aptamer), < 0.001, < < 0.05 against fibrillar GST-51Q-htt (in the absence of aptamer). (e) Densitometric analysis of the dots obtained (in Supplementary Figure S5c) was carried out with ImageQuantTL software (GE Healthcare). The ratio of intensities (of GAPDH-His6 sequestered by GST-51Q-htt with and without mHtt2.3.42) at the initial time point was assigned a value of 100%. The ratio of intensities at each time point is calculated with respect to this value. Values shown are mean ± SEM of three independent experiments.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Analysis of sequence similarity among RNA aptamers. (a) The randomized regions of RNA aptamers were aligned using Multalin software.49 (b) Structural similarities between mHtt2.2.47, mHtt2.2.18, and mHtt are shown. Secondary structures of the complete sequences (variable and conserved regions) were predicted by m-fold software.30 The values in parentheses below the figures show the free energy changes of the predicted structures at 37 °C. The consensus regions obtained using Multalin are shown for comparison.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Effect of intramers on solubility of 103Q-htt-mRFP in yeast cells. (a) Yeast cells were contransformed with constructs for expression of 103Q-htt-mRFP and aptamers as described in Methods section. Aggregation pattern was monitored by fluorescence microscopy. Cells expressing either 25Q-htt-mRFP or 103Q-htt-mRFP and empty vector pWHE601 (E.V.) or aptamers were analysed by fluorescence microscopy. Representative result with one RNA aptamer (mHtt2.2.47) is shown. Bar = 10 μm. (b) Native PAGE analysis of 103Q-htt-mRFP expressed in the presence of RNA aptamers in yeast cells (intramers). The gel was scanned with an image scanner (Typhoon Trio, GE Healthcare), using ℓex = 532 nm, ℓem = 610 nm. 103Q-htt-mRFP expressed in the presence of Lane 1: alone; Lane 2: mHtt2.2.17; Lane 3: mHtt2.2.18; Lane 4: mHtt2.2.25; Lane 5: mHtt2.2.47; Lane 6: mHtt2.3.08; Lane 7: mHtt2.3.21; Lane 8: mHtt2.3.24; Lane 9: mHtt2.3.42; Lane 10: mHtt2.3.43; Lane 11: mHtt2.4.08; Lane 12: empty vector (pWHE601); Lane 13: Saccharomyces cerevisiae BY4742 ▵Hsp104 strain. (c) The intensities of the bands for 103Q-htt-mRFP on native PAGE were quantified by densitometry (Image Quant TL, GE Healthcare). An arbitrary value of 1 was assigned to the band intensity for 103Q-htt-mRFP coexpressed with the empty vector. Values shown are mean ± SEM of three independent experiments. **P < 0.01, *P < 0.05 against the intensity of the band in cells coexpressing 103Q-htt-mRFP and empty vector (pWHE601). (d) Viability of yeast cells coexpressing 103Q-htt and noninhibitor (NI) or the indicated aptamer was determined by comparing colony forming units in each case. Values shown are mean ± SEM of three independent experiments. ***P < against cells coexpressing 103Q-htt and NI. (e) Extent of aggregation in cells coexpressing 103Q-htt and a pair of noninhibitors (NI+NI) or aptamers, as indicated, was monitored by solubilization with formic acid. Cell pellets were incubated in 100% formic acid for 30 minutes at 37 °C and diluted (1:150) in lysis buffer. Samples were centrifuged and supernatants obtained were filtered through a nitrocellulose membrane. The blot was probed with anti-polyglutamine antibody. A representative blot is shown. An arbitrary value of 100% was assigned to the intensity of dot obtained for 103Q-htt-EGFP coexpressed with a pair of noninhibitors (NI+NI). Values shown are mean ± SEM of three independent experiments. ***P < against cells coexpressing 103Q-htt and a pair of noninhibitors (NI+NI). (f) Measurement of oxidative stress in cells coexpressing 103Q-htt and a pair of noninhibitors (NI+NI) or aptamers, as indicated, using DCFH-DA. Values shown are mean ± SEM of three independent experiments. **P < 0.005, ***P < against cells coexpressing 103Q-htt and a pair of noninhibitors (NI+NI). (g) Viability of yeast cells coexpressing 103Q-htt and a pair of noninhibitors (NI+NI) or aptamers, as indicated, was determined by comparing colony forming units in each case. Values shown are mean ± SEM of three independent experiments. ***P < against cells coexpressing 103Q-htt and a pair of noninhibitors (NI+NI).
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
Aptamers rescue defect in endocytosis in yeast cells expressing 103Q-htt. Yeast cells were induced to express either 25Q-htt-EGFP or 103Q-htt-EGFP. (a) Endocytosis was monitored by internalization of the lipophilic dye FM4-64 after 5 minutes of incubation of the induced cells with the dye, as described in the Methods section. Internalization of the dye was monitored in cells expressing 25Q-htt or 103Q-htt-EGFP or in cells coexpressing 25Q-htt or 103Q-htt-EGFP and mHtt (inhibitor) or 103Q-htt and mHtt (noninhibitor); λex = BP 510–560 nm, λem = BA 590 nm for FM4-64. Bar = 10 μm. (b) Endocytosis was monitored by internalization of the lipophilic dye FM4-64 by yeast cells coexpressing 103Q-htt and mHtt (noninhibitor) after 10 (upper two panels) or 20 (lower two panels) minutes of incubation of the induced cells with the dye. (c) Comparison of internalization of FM4-64 in yeast cells expressing 25Q-htt-EGFP (empty bars) or 103Q-htt-EGFP (filled bars) in the presence or absence of inhibitor (I, mHtt2.2.47) or noninhibitor (NI, mHtt2.3.07) sequences after incubation for 5 or 20 minutes. 100% value on y-axis assumes that all the cells expressing either 25Q-htt or 103Q-htt-EGFP have internalized FM4-64. Twenty fields, each containing 40–50 cells, were counted in each case. Values represent mean ± SEM of three independent experiments. ***P < against cells expressing 25Q-htt-EGFP (without RNA sequence) incubated with the dye for the same time period; ###P < against cells expressing 103Q-htt-EGFP (without RNA sequence) incubated with the dye for the same time period; N.S., nonsignificant.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions