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Non-B Conformations of CAG Repeats Using 2-Aminopurine
C H A P T E R
S E V E N
Non-B Conformations of CAG Repeats Using 2-Aminopurine Natalya N. Degtyareva and Jeffrey T. Petty Contents 1. Introduction 1.1. Diseases associated with repeated sequences 1.2. 2-Aminopurine as a structural and energetic probe 2. Materials and Methods 2.1. Materials 2.2. Conformational integrity 2.3. Calculation of thermodynamic parameters 2.4. Oligonucleotide concentration 2.5. Structural references 2.6. Acrylamide quenching 3. Structure and Thermodynamics of Isolated and Integrated (CAG)8 3.1. (CAG)8 hairpin 3.2. (CAG)8 three-way junction 4. Conclusions Acknowledgments References
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Abstract Repetition of trinucleotide sequences is the molecular basis of 30 hereditary neurological and neurodegenerative diseases, and alternate structures adopted by these sequences are implicated in the etiology of such diseases. Elucidating these structures is important for advancing mechanistic understanding and ultimately treatment. Studies of (CAG) repeats are motivated by their involvement in a number of these diseases, and the structures favored by (CAG)8 are discussed in this contribution. Utilizing the strong effect of base stacking on fluorescence quantum yield, 2-aminopurine is used in place of adenine to determine the secondary structures adopted by such repeated sequences. Alone, (CAG)8 folds into a hairpin comprised of a duplex stem and a singlestranded loop. Energetic studies indicate that the hairpin is anchored by the interactions in the stem and has a strained loop environment. As a model for Department of Chemistry, Furman University, Greenville, South Carolina, USA Methods in Enzymology, Volume 492 ISSN 0076-6879, DOI: 10.1016/B978-0-12-381268-1.00019-7
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2011 Elsevier Inc. All rights reserved.
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intermediates that form during repeat expansion, (CAG)8 was also incorporated into a duplex to form a three-way junction. In contrast to the isolated (CAG)8, this integrated repeat adopts an open, unfolded loop. Enthalpy and entropy changes associated with denaturation indicate that the stability of the threeway junction is dominated by interactions in the duplex arms and that the repeated sequence tracks global unfolding. Because 2-aminopurine provides both structural and energetic information via fluorescence and also is an innocuous substitution for adenine, significant progress in elucidating the secondary structures of (CAG) repeats will be achieved.
1. Introduction 1.1. Diseases associated with repeated sequences Modifications in the genetic code can alter cellular function and ultimately lead to disease. Besides exogenous agents, mutagenicity can originate within DNA itself, and such genetic instability is illustrated by a class of 30 neurological and neurodegenerative diseases that are associated with repeated DNA sequences (Bacolla and Wells, 2009; Gatchel and Zoghbi, 2005; Kovtun and McMurray, 2008; Mirkin, 2007). Fragile X syndrome and other associated diseases are distinguished by their preponderance in humans and by their non Mendelian genetics in which the probability of mutation increases in subsequent generations. A critical insight was that sequence expansion beyond a threshold dictates development and severity of such diseases, and an underlying question is whether alternative secondary structures of these long DNA sequences are significant (Bacolla and Wells, 2009; Cleary and Pearson, 2003; Mirkin, 2007). Central to models for repeat expansion are self-associated forms of single-stranded DNA, such as the hairpins adopted by CAG and CTG repeats (Lenzmeier and Freudenreich, 2003; Sinden et al., 2007). These particular sequences are implicated in a large subset of the repeat-based neurological diseases, and their folding is driven by duplex stem formation (Amrane et al., 2005; Mariappan et al., 1998; Mitas et al., 1995; Zheng et al., 1996). The resulting mismatches of like bases destabilize the hairpin, but this instability is tempered by stacking with neighboring G/C pairs (Amrane et al., 2005; Mitas, 1997; Paiva and Sheardy, 2004). Expansion of repeated sequences occurs when one strand is preferentially extended relative to its complementary sequence during processes such as replication, so it is important to consider how DNA context influences the secondary structures of repeated sequences. One type of model structure is the slipped intermediate heteroduplex that has a singlestranded repeated sequence that emanates from the duplex (Pearson et al., 2002; Sinden et al., 2007). Using enzymatic probes, the key structural
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elements that have been identified are strand junctions, hairpin loops, and the core of the repeated sequences. While folding in CTG repeats is not influenced by their DNA context, folding in CAG repeats is disrupted in these heteroduplexes, and this structural difference may be important in the biological processing of these two types of repeats. Beyond enzymatic and chemical probing and relatively low-resolution microscopy techniques, high-resolution structural studies of repeated sequences are significantly advanced by using fluorogenic bases.
1.2. 2-Aminopurine as a structural and energetic probe Via changes in intensity, spectral position, lifetime, and anisotropy, fluorescence spectroscopy offers a sensitive spectroscopic perspective on both the structure and energetics of nucleic acids (Hawkins et al., 2008; Lakowicz, 2006). Low-fluorescence quantum yields of the natural nucleobases restrict such investigations, thus motivating development of fluorescent base analogs (Sinkeldam et al., 2010). In general, these modified bases have three features that furnish structural information at the base level. First, selective detection of the modified analogs is possible because their emission intensity and spectra are resolved from the natural nucleobases. Second, chemical synthesis allows precise placement of the analogs within the primary sequence of the nucleic acid. Third, spectra can depend on interactions such as base stacking and thus provide a convenient spectroscopic means to assess local structure within the overall structure. An important issue is the extent to which modified bases reflect and possibly influence DNA structure, and in this regard, 2-aminopurine has been extensively investigated (Rist and Marino, 2002). This isomer of adenine has the exocyclic amine group in the second versus the sixth position on adenine, and it maintains base pairing with thymine in B-form DNA ( Johnson et al., 2004; Lycksell et al., 1987; Sowers et al., 1986). A key feature of 2-aminopurine is its high fluorescence quantum yield of 50–70% under physiological conditions, and its value for nucleic acid structural studies is that the quantum yield is quenched 100-fold due to stacking interactions with adjacent bases (Rachofsky et al., 2001; Ward et al., 1969). Because secondary structural elements of nucleic acid structure depend on base stacking, this large variation in fluorescence quantum yield allows fine discrimination of structural features (Rist and Marino, 2002). The goal of our studies is to determine the structure of repeated (CAG) sequences using selective substitutions of adenine with 2-aminopurine, and two studies provided guidance. First, a constrained hairpin with a loop comprised of three (CAG) repeats showed that the spectral properties of the substituted 2-aminopurines depend on their location in the primary sequence (Lee et al., 2007). Variations in intensity and anisotropy suggest that base stacking and hydrogen bonding are significant factors in stabilizing the repeated sequences in the loop. Second, key structural and energetic
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features of RNA have been investigated using 2-aminopurine (Ballin et al., 2007, 2008). In these studies, extrahelical/bulged, base paired, and looped bases were identified by the fluorescence intensities and lifetimes of selectively substituted 2-aminopurines. These studies are distinguished by their use of control single-stranded sequences to isolate structural from temperature and base context effects on the fluorescence properties of 2-aminopurine. Our studies use these important contributions to understand the structures and thermodynamics of repeated (CAG) sequences.
2. Materials and Methods 2.1. Materials Oligonucleotides from Integrated DNA Technologies (Coralville, IA) were synthesized using standard phosphoramidite chemistry and purified using denaturing gel electrophoresis with 7 M urea and 8% polyacrylamide (Table 7.1). A 5-base standard was used to identify DNA strands based on their length, and the desired band was excised and then electrocuted from a dialysis bag. Following buffer exchange using gel filtration chromatography (NAP-10, GE Healthcare), the DNA was lyophilized and resuspended in buffer. Three-way junctions were formed by annealing equimolar quantities of 50- and 74-base oligonucleotides, with the longer DNA strand containing a central (CAG)8 with flanking 25-base regions that complement the shorter strand (Fig. 7.1). Measurements were conducted in buffers comprised of 10 mM Tris/TrisHþ (pH ¼ 7.9) with 10 mM MgCl2 and 50 mM NaCl or 10 mM H2PO4/HPO42 (pH ¼ 7) with 50 mM NaCl. The results show no buffer dependence. Nomenclature for modified oligonucleotides identifies the position of 2-aminopurine within the primary sequence (Fig. 7.1). Starting from the 50 end, individual replacements of adenine with 2-aminopurine in the isolated (CAG)8 hairpin were made in the first, second, third, fourth, fifth, and sixth repeats, and these are designated by 1-APHP, 2-APHP, 3-APHP, 4-APHP, 5-APHP, and 6-APHP, respectively. For the three-way junction, five substitutions notated as 1-APJ, 3-APJ, 4-APJ, 5-APJ, and 6-APJ were made in the first, third, fourth, fifth, and sixth repeats of the central (CAG)8, respectively. Modifications labeled a-APJ and b-APJ were also made in the two (CAG) repeats in the duplex region that precedes the junction.
2.2. Conformational integrity To evaluate how 2-aminopurine influences the secondary structure of repeated sequences, circular dichroism, thermal denaturation, gel electrophoresis, and enzyme recognition are used. In the ultraviolet spectral
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Table 7.1 Single-stranded oligonucleotides
a b c d e
Sequence
Lengtha eb
50 -CACCATGCCGGTA TTTAAA CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG TACGTA CTGCAGCTCGAGG-30 0 5 -CACCATGCC GGT ATT TAA ACAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG TACGTA CTGCAGCTCGAGG-30 c 50 -CCTCGAGCTGCAG TACGTA CTGCTG─CTGCTG TTTAAA TACCGGCATGGTG-30 d 0 5 -CACCATGCCGGTA TTTAAA CAGCAG CAGCAG TACGTA CT GCAGCTCGAGG-30 0 5 -CAG CAG CAG CAG CAG CAG CAG CAG-30 50 -CAG CAG CAG CAG CAG CAG CAG CAG-30 e 50 -G AAA CAG CAG TTTT CTG CTG TTT C-30 (DS-CAG) 50 -AAA CAG CAG-30 (SSb-CAG) 50 -CAG CAG CAG-30 (SS-CAG)
74
720,500
74
708,700
50
462,500
50
484,500
24 24 24
237,300 225,500 210,900
9 9
86,300 78,000
Length in bases. Extinction coefficients (M 1 cm 1) of the unfolded single strands. Seven variants of this oligonucleotide were used, each with a single substitution of 2-aminopurine, indicated by the underlined bases. Connecting line represents the region corresponding to the (CAG)8. Six different substitutions of 2-aminopurine were made in (CAG)8.
region, base–base interactions dominate the circular dichroism response to provide a sensitive indication of nucleic acid structure (Cantor and Schimmel, 1980). The hairpins adopted by (CAG) repeats have a conformation that is most similar to B-form DNA, which suggests that canonical G/C base pairs drive folding (Paiva and Sheardy, 2004). With respect to the unmodified sequences, similar circular dichroism spectra indicate that single 2-aminopurines throughout (CAG)8 have a minimal impact on conformation (Fig. 7.2). To gain an energetic perspective, melting temperatures, enthalpy changes, and entropy changes associated with global denaturation were derived from absorbance changes at 260 nm, and similar thermodynamic values with and without 2-aminopurine indicate that the stabilities of repeat structures are not perturbed by modification (Tables 7.2 and 7.3). To monitor DNA shape, nondenaturing gel electrophoresis shows that the mobility of (CAG)8, 24-base oligonucleotide is comparable to that of a 12 base pair duplex, and no changes with 2-aminopurine were observed (Degtyareva et al., 2009). For the three-way junction, only one band with a mobility that is distinct from its constituent single strands was observed (Degtyareva et al., 2010). Using restriction digestion at the two sites adjacent
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A
B
C
Figure 7.1 (A) Structure for the hairpin formed by (CAG)8. (B) Structure for the three-way junction formed with (CAG)8. Restriction enzyme sites for DraI and BsaAI are indicated in bold and underlined. Below this structure is the duplex that comprises the three-way junction. The connecting lines represent the position of the abstracted repeat sequence. (C) Single- and double-stranded oligonucleotides used as structural references. In all these oligonucleotides, the positions of 2-aminopurine substitution are underlined and/or enumerated.
to the strand junction, gel electrophoresis shows proper strand alignment by liberation of the terminal 16 base pair fragments. Again, 2-aminopurine does not influence these results.
2.3. Calculation of thermodynamic parameters Because thermal denaturation produces a common single-stranded state, differences in the thermodynamic parameters between the variants can be attributed to the low-temperature folded forms. Changes in the extinction coefficients of the bases and the fluorescence quantum yield of 2-aminopurine permit the folded and unfolded states to be defined, from which the energetic factors that impact global and local structure are deduced.
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(CAG)8 1-APHP
20
6-APHP 5-APHP 4-APHP
Ellipticity (m deg)
10
3-APHP 2-APHP
0
–10
–20 200
250
300 Wavelength (nm)
350
400
Figure 7.2 Circular dichroism spectra of unmodified and 2-aminopurine modified (CAG)8 sequences. The similarity in the spectra suggest that 2-aminopurine has no significant effect on the conformation of (CAG)8. Spectral shapes are consistent with a B-like duplex stem (Paiva and Sheardy, 2004).
Standard methods of extracting thermodynamic information are used to generate plots of fraction of folded species, y, versus temperature (Mergny and Lacroix, 2003). Different baselines corresponding to the low- and hightemperature states are used to estimate the uncertainties in the derived quantities. Melting temperatures are determined as the intersection point between the y dependence on temperature and the median between the two baselines. A more thorough thermodynamic analysis uses a two-state analysis to extract enthalpy and entropy changes after accounting for the molecularity of the transitions. Data was analyzed between y ¼ 0.2–0.8, where intermediate temperatures give relatively comparable concentrations of both folded and unfolded forms. Changes in entropy and enthalpy were measured for triplicate samples.
2.4. Oligonucleotide concentration Structural references are used to deduce the secondary structures of repeated sequences, and such comparisons must consider oligonucleotide concentrations. A sensitive method utilizes the high fluorescence quantum yield of unconjugated 2-aminopurine, and the exonuclease BAL-31 cleaves the fluorogenic nucleotide from the oligonucleotide. Concomitant base unstacking in dilute solution results is a significant enhancement in
Table 7.2 Thermodynamic data for (CAG)8 hairpin
a b c d
Modification
Tm ( C)a
DH (kcal/mol)a
DS (cal/mol/K)a
Tm ( C)b
DH (kcal/mol)b
DS (cal/mol/K)b
Kq (M 1)
1-APHP 2-APHP 3-APHP 4-APHP 5-APHP 6-APHP Unmodified SS-CAG DS-CAG
58.8 (1.0)d 60.1 (1.4) 60.3 (1.0) 61.1 (1.0) 59.2 (1.4) 60.2 (1.0) 57.4 (0.6)
32.0 (1.9) 28.5 (2.4) 29.9 (4.7) 32.1 (1.4) 28.9 (1.5) 29.5 (3.7) 29.0 (1.5)
96 (6) 86 (8) 90 (14) 96 (4) 87 (5) 88 (11) 88 (5)
NDc 52.8 (0.3) 54.4 (0.9) 50.4 (0.6) 47.0 (1.0) 51.8 (0.3)
ND 31.2 (0.9) 32.7 (1.3) 32.9 (2.9) 20.0 (2.0) 32.7 (1.3)
ND 96 (3) 100 (4) 95 (9) 61 (6) 100 (4)
9.8 (0.5) 5.0 (0.5) 9.1 (0.8) 11.8 (1.0) 14.8 (1.2) 10.6 (0.4)
Data derived from absorbance changes at 260 nm. Data derived from fluorescence changes of 2-aminopurine modified forms of (CAG)8. Values not determined. Standard deviations in parentheses derived from three measurements.
9.6 (1.0) 8.2 (0.6)
Table 7.3 Thermodynamic data for (CAG)8 three-way junction
a b c d
Modification
Tm ( C)a
DH (kcal/mol)a
DS (cal/mol/K)a
Tm ( C)b
DH (kcal/mol)b
DS (cal/mol/K)b
Kq (M 1)
b-APJ a-APJ 1-APJ 3-APJ 4-APJ 5-APJ 6-APJ Unmodified Duplex
75.9 (0.4)d 77.2 (0.1) 76.6 (0.1) 76.8 (0.8) 76.4 (0.1) 75.9 (0.3) 76.4 (0.6) 77.2 (0.2) 81.4 (0.3)
220 (22) 199 (20) NDc ND 194 (19) ND 217 (22) 199 (20) 243 (25)
606 (61) 543 (54) ND ND 522 (53) ND 600 (60) 540 (54) 665 (67)
77.0 (0.6) 78.7 (0.3) ND 77.0 (1.7) 77.1 (0.6) 77.6 (1.6) 76.5 (0.3)
238 (24) 237 (32) ND 224 (22) 190 (20) ND 250 (25)
630 (63) 650 (65) ND 606 (61) 516 (52) ND 685 (69)
7.5 (0.2) 9.8 (0.5) 11.1 (0.2) 15.0 (0.2) 13.5 (0.7) 15.2 (0.7) 14.9 (0.4)
Data derived from absorbance changes at 260 nm. Data derived from fluorescence changes of 2-aminopurine modified forms of the three-way junctions. Values not determined. Standard deviations in parentheses derived from three measurements.
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fluorescence intensity, which is then related to concentration using a standard curve. Typically, 5–20 nmol of DNA (in base pairs) were mixed with 0.5 units of BAL-31 in digestion buffer (20 mM Tris–HCl, pH 8.0, 600 mM NaCl, 12 mM CaCl2, 12 mM MgCl2, 1 mM EDTA) in a total volume of 50 mL and incubated at 30 C for 20–30 min. The reaction mixture was then diluted with buffer, and fluorescence intensities of the standard and digested samples were measured. Alternatively, because the sequences of the oligonucleotides are known, concentrations were also calculated using extinction coefficients for the cleaved nucleotides (Cavaluzzi and Borer, 2004). Finally, concentrations of intact oligonucleotides were determined using extinction coefficients at 260 nm derived from the nearest-neighbor approximation, with the small contribution from 2-aminopurine (1000 M 1 cm 1 at 260 nm) included in the calculation (Fox et al., 1958). To account for the effect of temperature, high-temperature posttransition baselines were extrapolated back to 25 C. Variations in DNA concentration determined by all three methods were less than 10%.
2.5. Structural references To relate the fluorescence from the 2-aminopurine labeled repeated sequences to particular secondary structural elements of DNA, single- and doublestranded references are used (Ballin et al., 2007, 2008) (Fig. 7.1). Nine-base oligonucleotides provide the environments for 2-aminopurine in solventexposed, single-stranded DNA, and a 2-aminopurine/thymine base pair in a duplex stem provides a reference for the fluorophore in a base-stacked and solvent-sequestered state. Although structurally analogous to the adenine/ thymine pair, the 2-aminopurine/thymine pair has faster proton exchange rates with the solvent and lower thermal stability, indicative of less effective stacking within the duplex (Lycksell et al., 1987; Patel et al., 1992; Xu et al., 1994). In both reference oligonucleotides, the immediate base environment of the 2-aminopurine is identical to that in the repeated sequence. In addition to secondary structure and neighboring bases, temperature also influences the fluorescence quantum yield of 2-aminopurine (Fig. 7.3). To dissect these contributions during thermal denaturation, the ratios of fluorescence intensities from the single-stranded reference and repeated sequences are used, as the former maintain an unfolded state over the entire temperature range. At high temperatures, similar fluorescence intensities indicate that both types of DNA achieve the same unfolded state (Figs. 7.3 and 7.4).
2.6. Acrylamide quenching To complement information derived from the inherent fluorescence of 2-aminopurine, extrinsic fluorescence quenching by acrylamide is also used to assess solvent exposure (Lakowicz, 2006). By relating quenching
223
Normalized intensity
2-Aminopurine Studies of CAG Repeats
1.4 × 106
Emission intensity (cps)
1.2
1.2 0.8 0.4 0.0
1.0
20 40 60 80 Temperature (⬚C)
0.8
0.6
0.4 20
30
40 50 Temperature (⬚C)
60
70
80
Figure 7.3 Emission intensities of 3-APHP (dashed) and SS-CAG (solid) as a function of temperature. The intensity change associated with the single-stranded reference is due solely to temperature, as this oligonucleotide is unfolded over the entire temperature range. The inset shows the normalized intensity for 3-APHP using the reference to account for temperature effects on the emission intensity. Because of denaturation of the (CAG)8 hairpin at high temperature, both oligonucleotides achieve comparable intensities.
efficiency to secondary structural elements of DNA, structural variations within the oligonucleotides are inferred (Ballin et al., 2007, 2008). Standard Stern–Volmer analysis is used to extract quenching constants, Kq, for the 2-aminopurines (Fig. 7.5, Tables 7.2 and 7.3). The smallest quenching constants are associated with bases that are most protected from the solvent, as in a duplex. As the fluorogenic base becomes more exposed to the aqueous solution, as for single-stranded DNA, the quenching constant increases.
3. Structure and Thermodynamics of Isolated and Integrated (CAG)8 3.1. (CAG)8 hairpin The overall conclusion from 2-aminopurine studies of (CAG)8 alone is that this repeated sequence favors a hairpin displaying suppressed fluorescence in the stem and enhanced fluorescence in the loop (Figs. 7.1 and 7.4). Folding driven by strand association is indicated by the similar behaviors of the distant modifications 2-APHP/3-APHP and 6-APHP. These three variants exhibit relatively lower emission intensities when compared to single-stranded
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Relative emission intensity
3.5
5-APHP
3.0 G
2.5
6-APHP
2.0 Relative emission intensity
Stem
1.0 3-AP HP 2-APHP
1.6 1.2
10
A*
C
G
G A*
C A
C
G
G
C A
A* C
DS-CAG
0
C
A*
1.5 4-APHP
0.0
A* Loop G
C G
2.0 1-APHP
0.5
C
A*
20
30 40 50 60 Temperature (ºC)
70
80
G
90
6-APJ 4-APJ 3-APJ 5-APJ
A*
G
C
A*
1-APJ
C
A*
A* G
C
0.8
a–APJ
G
C G
0.4
A* C Duplex G C A* G C A* G T C G T C
0.0 40
C A
G A
b–APJ
50
60
70
80
Loop
G
C G
C A G C A G C A G C G T C
G T
90
Temperature (ºC)
Figure 7.4 (Top) Emission intensities of 2-aminopurine-modified (CAG)8 as a function of temperature normalized using the emission from SS-CAG (lex ¼ 307 nm, lem ¼ 370 nm). (Bottom) Normalized emission intensities of the modified three-way junctions as a function of temperature. To the right of each graph are the models for the secondary structure based on fluorescence intensities. The graphic emphasis at each position indicates its level of solvent exposure.
DNA, and these intensities are comparable at high temperature. These two observation support base stacking in the folded, low-temperature form of (CAG)8 and are consistent with the propensity of repeated sequences to form into hairpins with B-like conformations in the stem (Paiva and Sheardy, 2004). Incorporation of the resulting purine–purine mismatches in the stem of (CAG)8 is gauged using a duplex with a 2-aminopurine/thymine base pair. Comparable intensities for matched and mismatched pairs involving 2-aminopurine indicate their comparable degrees of stacking with neighboring bases, a conclusion that is supported by NMR studies of duplexes containing adenine/adenine mismatches (Mariappan et al., 1998). The other major structural element identified by 2-aminopurine is a loop between the duplex. Relative to the single-stranded reference, 5-APHP has a higher
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2-Aminopurine Studies of CAG Repeats
5-APHP
3.0
6-APHP 4-APHP
2.5
F0 / F
SS-CAG 3-APHP
2.0
1-APHP DS-CAG
1.5
2-APHP 1.0 0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
Acrylamide concentration (M) 5-APJ
3.0
6-APJ 3-APJ 2.5
4-APJ
F0 / F
1-APJ a –APJ
2.0
SS-CAG b –APJ
1.5
DS-CAG
1.0 0.00
0.02
0.04
0.06
0.08
0.10
0.12
Acrylamide concentration (M)
Figure 7.5 Stern–Volmer plots of the acrylamide quenching of 2-aminopurine variants of (CAG)8 (top) and the three-way junction (bottom). F0 and F are the fluorescence intensities in the absence and presence of acrylamide, respectively. Linear fits were used to derive quenching constants using the standard Stern–Volmer model.
intensity that diminishes with temperature. This is consistent with a constrained loop that relaxes to enhance base stacking and the associated solvent sequestration in the single-stranded form. An important advantage of
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2-aminopurine is the strong effect of base stacking on fluorescence quantum yield, thereby resolving finer details of secondary structure. For (CAG)8, transitional behavior between the stem and loop motifs is evident. For 1APHP, its intensity is comparable to the single-stranded reference over the entire temperature range, consistent with frayed, unpaired terminal repeats. Features of both the stem and loop are exhibited by 4-APHP, which has an intermediate fluorescence intensity and smaller fluorescence enhancement. This behavior is considered in the context of models of repeat folding, which indicate that stacking interactions in the stem are maximized by the tetraloop AGCA (Fig. 7.1; Hartenstine et al., 2000). In (CAG)8, these bases comprise the fourth and fifth repeats, yet variations in the fluorescence properties of 2-aminopurine substitutions in these repeats suggest an asymmetric loop environment. Local variations in the interactions with neighboring bases could be dictating the structure of the loop (Mariappan et al., 1996; Senior et al., 1988; Varani, 1995). Structural patterns deduced from fluorescence are supported by acrylamide quenching studies, which show clustering into three groups (Fig. 7.5, Table 7.2). The least efficient quenching is observed for 2-APHP and the duplex reference, which supports protection for 2-aminopurine within a duplex stem from this external quenching agent. Higher solvent exposure comparable to the single-stranded reference is observed for 1-APHP, 3-APHP, 4-APHP, and 6-APHP. While the frayed ends of 1-APHP and the single-stranded reference are expected to be structurally similar, the fluorescence properties of 3-APHP, 4-APHP, and 6-APHP indicate that these 2-aminopurine substitutions are more solvent sequestered. Thus, these similarities suggest that acrylamide quenching constants should serve as general guides to the structures of repeated sequences. The largest quenching constant is observed for 5-APHP, consistent with the high fluorescence intensity from this region of the loop. Quenching is as efficient as observed for 2-aminopurine at the exposed bulge of a RNA duplex, but is smaller than that of the free nucleobase (Ballin et al., 2007; Degtyareva et al., 2009). As utilized in structural studies, denaturation of (CAG)8 produces a single-stranded oligonucleotide, so high temperature fluorescence spectra provide an important structural reference. Furthermore, temperaturedependent fluorescence changes allow the fractional conversion from folded to unfolded states to be derived, from which enthalpy and entropy changes are determined using Arrhenius analysis (Table 7.2). A context for interpreting local energetic changes is provided by global unfolding using hyperchromic absorbance changes at 260 nm. These enthalpy and entropy changes are similar to values obtained from calorimetric analysis, which suggests that neglecting heat capacity changes does not significantly compromise interpretation of the results (Amrane et al., 2005; Paiva and Sheardy, 2004). For 2-APHP, 3-APHP, and 6-APHP, local enthalpy and entropy changes derived from fluorescence are similar to the global values.
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This consistency suggests that base pairing and stacking the stem dictate the overall stability of the folded hairpin. In contrast, 5-APHP has both smaller enthalpy and entropy changes for denaturation relative to the global values. These energetic features complement the relatively high fluorescence from this region of the hairpin and support limited base interactions in the loop of the hairpin. Thermodynamic measurements also provide insight into how the mismatch influences the stability of the stem. Global melting temperatures derived from absorbance are 6–8 C higher than those derived from fluorescence. This behavior suggests that neighboring base interactions with 2-aminopurine weaken and thereby enhance solvent exposure prior to global denaturation (Lycksell et al., 1987; Nordlund et al., 1993).
3.2. (CAG)8 three-way junction Fluorescence from 2-aminopurine shows that integration of (CAG)8 into a duplex disrupts intrastrand association within the repeat sequence to produce an open and solvent-exposed loop (Figs. 7.1 and 7.4). To understand this context-dependent structural change for (CAG)8, seven modifications were used to map solvent exposure throughout the three-way junction. Within the duplex arms, (CAG) repeats base pair with opposing (GTC) repeats in the complementary strands, as shown by b-APJ and a-APJ. Emission intensities from these variants are lower than the single-stranded reference and increase with denaturation at high temperature, and both observations support base stacking of 2-aminopurine that is relieved with thermal denaturation. However, the strand junction perturbs base pairing and stacking within the duplex, as indicated by the higher intensities of b-APJ and a-APJ relative to the duplex reference with a 2-aminopurine/ thymine base pair. Furthermore, a-APJ has a higher intensity than b-APJ, suggesting that the level of solvent exposure increases with proximity to the junction. This trend in solvent exposure continues into the repeated (CAG)8 sequence, with 1-APJ acting as single-stranded DNA over the entire temperature range. Collectively, the fluorescence properties of these three modifications indicate that the transition from duplex to repeated sequence is accompanied by disrupted base interactions that increase solvent exposure of the bases. This disturbance appears to be transmitted into the repeated sequence, with 3-APJ, 4-APJ, 5-APJ, and 6-APJ exhibiting similarly high fluorescence intensities when compared with the single-stranded reference. In addition, mirroring the loop region of the (CAG)8 hairpin, these four variants of the three-way junction display solvent exposure that diminishes at higher temperatures, suggesting that constraints induced by the junction relax when the three-way junction denatures. This openness of the repeated sequence strikingly contrasts with the folded (CAG)8, as most clearly demonstrated by the third and sixth repeats. Hairpin formation in (CAG)8 alone is shown by comparable
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and relatively low levels of solvent exposure in 3-APHP and 6-APHP. In contrast, similarly placed 2-aminopurines in 3-APJ and 6-APJ are more solvent exposed when compared with the single-stranded reference. Acrylamide quenching studies support a model in which solvent exposure becomes comparable to single-stranded DNA in the vicinity of the strand junction and is consistently high in the core of the repeated sequence (Fig. 7.5, Table 7.3). Quenching for b-APJ and the duplex reference are comparable, which suggests that the b modification is protected from extrinsic quenching by base stacking and pairing. Quenching becomes increasingly efficient proceeding toward the junction from a-APJ and onto 1-APJ. Quenching constants that are consistently higher than singlestranded DNA are observed for 3-APJ, 4-APJ, 5-APJ, and 6-APJ. Additional support for the open conformation throughout the integrated (CAG)8 is provided by the similarity of these quenching constants when compared with 5-APHP in the loop of the (CAG)8 hairpin. To understand the thermodynamic factors that contribute to the stability of the three-way junction, thermal denaturation was followed using absorbance and fluorescence spectral changes (Table 7.3). After dissecting the repeated sequence, the resulting duplex is shown to dominate the stability of the three-way junction. Specifically, enthalpy and entropy changes for the three-way junction are slightly ( 20%) lower than the values for the duplex. Furthermore, the melting profile of the three-way junction is monophasic with no evidence of a transition associated with the (CAG)8 hairpin. Local unfolding was monitored using the fluorescent variants, and the similarities of the melting temperatures, enthalpy changes, and entropy changes relative to the values derived from absorbance indicate that local structural changes track global unfolding. Collectively, these observations suggest that the stability of the three-way junction is dictated by its duplex arms with slight instability induced by the repeated sequence. The overall conclusion is that DNA context critically impacts the structure of (CAG)8. Enzymatic probes also highlight the distinctive features of (CAG) repeats when compared with (CTG) repeats (Pearson et al., 2002). In slipped intermediates, enzymatic recognition of the latter repeats is consistent with their folded, hairpin structures that also form in the analogous isolated repeats. In contrast, (CAG) repeats preferentially interact with single-strand specific enzymes and proteins, indicative of their random coil conformation. Such a conformation differs from the folded and largely protected hairpins favored by isolated (CAG) hairpins. This behavior again supports the dominant effect of the duplex arms and the strand junction on the structure of such repeated sequences. The significance of these earlier results and our studies using 2-aminopurine lies in the importance of secondary structure in the biological recognition and processing of repeated sequences via enzyme recognition (Kovtun and McMurray, 2008; Pearson et al., 1997).
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4. Conclusions The adenine isomer 2-aminopurine provides an avenue to understand the secondary structures of repeated DNA sequences, and a profound difference is noted when (CAG)8 is isolated from versus integrated into a duplex of canonical base pairs. Alone, (CAG)8 forms a hairpin in which folding is driven by duplex formation in the stem. In contrast, such distant intrastrand interactions are absent when (CAG)8 is incorporated into a duplex. This structural distinction suggests that the duplex has a significant effect on the structures of repeated sequences. These results provide the foundation for understanding the secondary structures favored by larger repeated sequences that are implicated in debilitating neurological diseases. Such structures will be elucidated from the repeat level resolution of structural and energetic information that is gleaned from 2-aminopurine. By identifying key structural elements favored by repeated sequences, the path to understanding the mechanisms of repeat expansion will have a stronger molecular basis.
ACKNOWLEDGMENTS We are grateful to the National Science Foundation (CHE-0718588) and the Henry Dreyfus Teacher–Scholar Awards Program for the primary support for this work. We also greatly appreciate partial support from the National Science Foundation (CBET-0853692) and the National Institutes of Health (R15GM071370 and P20 RR-016461 (from the National Center for Research Resource)).
REFERENCES Amrane, S., Sacca, B., Mills, M., Chauhan, M., Klump, H. H., and Mergny, J.-L. (2005). Length-dependent energetics of (CTG)n and (CAG)n trinucleotide repeats. Nucleic Acids Res. 33, 4065–4077. Bacolla, A., and Wells, R. D. (2009). Non-B DNA conformations as determinants of mutagenesis and human disease. Mol. Carcinog. 48, 273–285. Ballin, J. D., Bharill, S., Fialcowitz-White, E. J., Gryczynski, I., Gryczynski, Z., and Wilson, G. M. (2007). Site-specific variations in RNA folding thermodynamics visualized by 2-aminopurine fluorescence. Biochemistry 46, 13948–13960. Ballin, J. D., Prevas, J. P., Bharill, S., Gryczynski, I., Gryczynski, Z., and Wilson, G. M. (2008). Local RNA conformational dynamics revealed by 2-aminopurine solvent accessibility. Biochemistry 47, 7043–7052. Cantor, C. R., and Schimmel, P. R. (1980). Biophysical Chemistry. First ed., W. H. Freeman and Company, New York. Cavaluzzi, M. J., and Borer, P. N. (2004). Revised UV extinction coefficients for nucleoside5’-monophosphates and unpaired DNA and RNA. Nucleic Acids Res. 32, e13.
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Cleary, J. D., and Pearson, C. E. (2003). The contribution of cis-elements to diseaseassociated repeat instability: Clinical and experimental evidence. Cytogenet. Genome Res. 100, 25–55. Degtyareva, N. N., Reddish, M. J., Sengupta, B., and Petty, J. T. (2009). Structural studies of a trinucleotide repeat sequence using 2-aminopurine. Biochemistry 48, 2340–2346. Degtyareva, N. N., Barber, C. A., Sengupta, B., and Petty, J. T. (2010). Context dependence of trinucleotide repeat structures. Biochemistry 49, 3024–3030. Fox, J. J., Wempen, I., Hampton, A., and Doerr, I. L. (1958). Thiation of nucleosides. I. Synthesis of 2-amino-6-mercapto-9-b-D-ribofuranosylpurine (“Thioguanosine”) and related purine nucleosides1. J. Am. Chem. Soc. 80, 1669–1675. Gatchel, J. R., and Zoghbi, H. Y. (2005). Diseases of unstable repeat expansion: Mechanisms and common principles. Nat. Rev. Genet. 6, 743–755. Hartenstine, M. J., Goodman, M. F., and Petruska, J. (2000). Base stacking and even/odd behavior of hairpin loops in DNA triplet repeat slippage and expansion with DNA polymerase. J. Biol. Chem. 275, 18382–18390. Hawkins, M. E., Ludwig, B., and Michael, L. J. (2008). Fluorescent pteridine probes for nucleic acid analysis. Methods in Enzymology Vol. 450, pp. 201–231. Academic Press, San Diego, CA, Chapter 10. Johnson, N. P., Baase, W. A., and von Hippel, P. H. (2004). Low-energy circular dichroism of 2-aminopurine dinucleotide as a probe of local conformation of DNA and RNA. Proc. Natl. Acad. Sci. USA 101, 3426–3431. Kovtun, I. V., and McMurray, C. T. (2008). Features of trinucleotide repeat instability in vivo. Cell Res. 18, 198–213. Lakowicz, J. R. (2006). Principles of Fluorescence Spectroscopy, Third ed., Springer, New York. Lee, B. J., Barch, M., Castner, E. W., Volker, J., and Breslauer, K. J. (2007). Structure and dynamics in DNA looped domains: CAG triplet repeat sequence dynamics probed by 2-aminopurine fluorescence. Biochemistry 46, 10756–10766. Lenzmeier, B. A., and Freudenreich, C. H. (2003). Trinucleotide repeat instability: A hairpin curve at the crossroads of replication, recombination, and repair. Cytogenet. Genome Res. 100, 7–24. Lycksell, P. O., Graslund, A., Claesens, F., McLaughlin, L. W., Larsson, U., and Rigler, R. (1987). Base pair opening dynamics of a 2-aminopurine substituted Eco RI restriction sequence and its unsubstituted counterpart in oligonucleotides. Nucleic Acids Res. 15, 9011–9025. Mariappan, S. V., Garcoa, A. E., and Gupta, G. (1996). Structure and dynamics of the DNA hairpins formed by tandemly repeated CTG triplets associated with myotonic dystrophy. Nucleic Acids Res. 24, 775–783. Mariappan, S. V., Silks, L. A., 3rd, Chen, X., Springer, P. A., Wu, R., Moyzis, R. K., Bradbury, E. M., Garcia, A. E., and Gupta, G. (1998). Solution structures of the Huntington’s disease DNA triplets, (CAG)n. J. Biomol. Struct. Dyn. 15, 723–744. Mergny, J.-L., and Lacroix, L. (2003). Analysis of thermal melting curves. Oligonucleotides 13, 515–537. Mirkin, S. M. (2007). Expandable DNA repeats and human disease. Nature 447, 932–940. Mitas, M. (1997). Trinucleotide repeats associated with human disease. Nucleic Acids Res. 25, 2245–2254. Mitas, M., Yu, A., Dill, J., Kamp, T. J., Chambers, E. J., and Haworth, I. S. (1995). Hairpin properties of single-stranded DNA containing a GC-rich triplet repeat: (CTG)15. Nucleic Acids Res. 23, 1050–1059. Nordlund, T. M., Xu, D., and Evans, K. O. (1993). Excitation energy transfer in DNA: Duplex melting and transfer from normal bases to 2-aminopurine. Biochemistry 32, 12090–12095.
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Paiva, A. M., and Sheardy, R. D. (2004). Influence of sequence context and length on the structure and stability of triplet repeat DNA oligomers. Biochemistry 43, 14218–14227. Patel, N., Berglund, H., Nilsson, L., Rigler, R., McLaughlin, L. W., and Gra¨slund, A. (1992). Thermodynamics of interaction of a fluorescent DNA oligomer with the anti-tumour drug netropsin. Eur. J. Biochem. 203, 361–367. Pearson, C., Ewel, A., Acharya, S., Fishel, R., and Sinden, R. (1997). Human MSH2 binds to trinucleotide repeat DNA structures associated with neurodegenerative diseases. Hum. Mol. Genet. 6, 1117–1123. Pearson, C. E., Tam, M., Wang, Y.-H., Montgomery, S. E., Dar, A. C., Cleary, J. D., and Nichol, K. (2002). Slipped-strand DNAs formed by long (CAG){middle dot}(CTG) repeats: Slipped-out repeats and slip-out junctions. Nucleic Acids Res. 30, 4534–4547. Rachofsky, E. L., Osman, R., and Ross, J. B. (2001). Probing structure and dynamics of DNA with 2-aminopurine: Effects of local environment on fluorescence. Biochemistry 40, 946–956. Rist, M. J., and Marino, J. P. (2002). Fluorescent nucleotide base analogs as probes of nucleic acid structure, dynamics and interactions. Curr. Org. Chem. 6, 775. Senior, M. M., Jones, R. A., and Breslauer, K. J. (1988). Influence of loop residues on the relative stabilities of DNA hairpin structures. Proc. Natl. Acad. Sci. USA 85, 6242–6246. Sinden, R. R., Pytlos-Sinden, M. J., and Potaman, V. N. (2007). Slipped strand DNA structures. Front. Biosci. 12, 4788–4799. Sinkeldam, R. W., Greco, N. J., and Tor, Y. (2010). Fluorescent analogs of biomolecular building blocks: Design, properties, and applications. Chem. Rev. 110, 2579–2619. Sowers, L. C., Fazakerley, G. V., Eritja, R., Kaplan, B. E., and Goodman, M. F. (1986). Base pairing and mutagenesis: Observation of a protonated base pair between 2-aminopurine and cytosine in an oligonucleotide by proton NMR. Proc. Natl. Acad. Sci. USA 83, 5434–5438. Varani, G. (1995). Exceptionally stable nucleic acid hairpins. Annu. Rev. Biophys. Biomol. Struct. 24, 379–404. Ward, D. C., Reich, E., and Stryer, L. (1969). Fluorescence studies of nucleotides and polynucleotides. I. Formycin, 2-aminopurine riboside, 2, 6-diaminopurine riboside, and their derivatives. J. Biol. Chem. 244, 1228–1237. Xu, D., Evans, K. O., and Nordlund, T. M. (1994). Melting and premelting transitions of an oligomer measured by DNA base fluorescence and absorption. Biochemistry 33, 9592–9599. Zheng, M., Huang, X., Smith, G. K., Yang, X., and Gao, X. (1996). Genetically unstable CXG repeats are structurally dynamic and have a high propensity for folding NMR UV spectroscopic study. J. Mol. Biol. 264, 323–336.
S E V E N
Non-B Conformations of CAG Repeats Using 2-Aminopurine Natalya N. Degtyareva and Jeffrey T. Petty Contents 1. Introduction 1.1. Diseases associated with repeated sequences 1.2. 2-Aminopurine as a structural and energetic probe 2. Materials and Methods 2.1. Materials 2.2. Conformational integrity 2.3. Calculation of thermodynamic parameters 2.4. Oligonucleotide concentration 2.5. Structural references 2.6. Acrylamide quenching 3. Structure and Thermodynamics of Isolated and Integrated (CAG)8 3.1. (CAG)8 hairpin 3.2. (CAG)8 three-way junction 4. Conclusions Acknowledgments References
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Abstract Repetition of trinucleotide sequences is the molecular basis of 30 hereditary neurological and neurodegenerative diseases, and alternate structures adopted by these sequences are implicated in the etiology of such diseases. Elucidating these structures is important for advancing mechanistic understanding and ultimately treatment. Studies of (CAG) repeats are motivated by their involvement in a number of these diseases, and the structures favored by (CAG)8 are discussed in this contribution. Utilizing the strong effect of base stacking on fluorescence quantum yield, 2-aminopurine is used in place of adenine to determine the secondary structures adopted by such repeated sequences. Alone, (CAG)8 folds into a hairpin comprised of a duplex stem and a singlestranded loop. Energetic studies indicate that the hairpin is anchored by the interactions in the stem and has a strained loop environment. As a model for Department of Chemistry, Furman University, Greenville, South Carolina, USA Methods in Enzymology, Volume 492 ISSN 0076-6879, DOI: 10.1016/B978-0-12-381268-1.00019-7
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2011 Elsevier Inc. All rights reserved.
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intermediates that form during repeat expansion, (CAG)8 was also incorporated into a duplex to form a three-way junction. In contrast to the isolated (CAG)8, this integrated repeat adopts an open, unfolded loop. Enthalpy and entropy changes associated with denaturation indicate that the stability of the threeway junction is dominated by interactions in the duplex arms and that the repeated sequence tracks global unfolding. Because 2-aminopurine provides both structural and energetic information via fluorescence and also is an innocuous substitution for adenine, significant progress in elucidating the secondary structures of (CAG) repeats will be achieved.
1. Introduction 1.1. Diseases associated with repeated sequences Modifications in the genetic code can alter cellular function and ultimately lead to disease. Besides exogenous agents, mutagenicity can originate within DNA itself, and such genetic instability is illustrated by a class of 30 neurological and neurodegenerative diseases that are associated with repeated DNA sequences (Bacolla and Wells, 2009; Gatchel and Zoghbi, 2005; Kovtun and McMurray, 2008; Mirkin, 2007). Fragile X syndrome and other associated diseases are distinguished by their preponderance in humans and by their non Mendelian genetics in which the probability of mutation increases in subsequent generations. A critical insight was that sequence expansion beyond a threshold dictates development and severity of such diseases, and an underlying question is whether alternative secondary structures of these long DNA sequences are significant (Bacolla and Wells, 2009; Cleary and Pearson, 2003; Mirkin, 2007). Central to models for repeat expansion are self-associated forms of single-stranded DNA, such as the hairpins adopted by CAG and CTG repeats (Lenzmeier and Freudenreich, 2003; Sinden et al., 2007). These particular sequences are implicated in a large subset of the repeat-based neurological diseases, and their folding is driven by duplex stem formation (Amrane et al., 2005; Mariappan et al., 1998; Mitas et al., 1995; Zheng et al., 1996). The resulting mismatches of like bases destabilize the hairpin, but this instability is tempered by stacking with neighboring G/C pairs (Amrane et al., 2005; Mitas, 1997; Paiva and Sheardy, 2004). Expansion of repeated sequences occurs when one strand is preferentially extended relative to its complementary sequence during processes such as replication, so it is important to consider how DNA context influences the secondary structures of repeated sequences. One type of model structure is the slipped intermediate heteroduplex that has a singlestranded repeated sequence that emanates from the duplex (Pearson et al., 2002; Sinden et al., 2007). Using enzymatic probes, the key structural
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elements that have been identified are strand junctions, hairpin loops, and the core of the repeated sequences. While folding in CTG repeats is not influenced by their DNA context, folding in CAG repeats is disrupted in these heteroduplexes, and this structural difference may be important in the biological processing of these two types of repeats. Beyond enzymatic and chemical probing and relatively low-resolution microscopy techniques, high-resolution structural studies of repeated sequences are significantly advanced by using fluorogenic bases.
1.2. 2-Aminopurine as a structural and energetic probe Via changes in intensity, spectral position, lifetime, and anisotropy, fluorescence spectroscopy offers a sensitive spectroscopic perspective on both the structure and energetics of nucleic acids (Hawkins et al., 2008; Lakowicz, 2006). Low-fluorescence quantum yields of the natural nucleobases restrict such investigations, thus motivating development of fluorescent base analogs (Sinkeldam et al., 2010). In general, these modified bases have three features that furnish structural information at the base level. First, selective detection of the modified analogs is possible because their emission intensity and spectra are resolved from the natural nucleobases. Second, chemical synthesis allows precise placement of the analogs within the primary sequence of the nucleic acid. Third, spectra can depend on interactions such as base stacking and thus provide a convenient spectroscopic means to assess local structure within the overall structure. An important issue is the extent to which modified bases reflect and possibly influence DNA structure, and in this regard, 2-aminopurine has been extensively investigated (Rist and Marino, 2002). This isomer of adenine has the exocyclic amine group in the second versus the sixth position on adenine, and it maintains base pairing with thymine in B-form DNA ( Johnson et al., 2004; Lycksell et al., 1987; Sowers et al., 1986). A key feature of 2-aminopurine is its high fluorescence quantum yield of 50–70% under physiological conditions, and its value for nucleic acid structural studies is that the quantum yield is quenched 100-fold due to stacking interactions with adjacent bases (Rachofsky et al., 2001; Ward et al., 1969). Because secondary structural elements of nucleic acid structure depend on base stacking, this large variation in fluorescence quantum yield allows fine discrimination of structural features (Rist and Marino, 2002). The goal of our studies is to determine the structure of repeated (CAG) sequences using selective substitutions of adenine with 2-aminopurine, and two studies provided guidance. First, a constrained hairpin with a loop comprised of three (CAG) repeats showed that the spectral properties of the substituted 2-aminopurines depend on their location in the primary sequence (Lee et al., 2007). Variations in intensity and anisotropy suggest that base stacking and hydrogen bonding are significant factors in stabilizing the repeated sequences in the loop. Second, key structural and energetic
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features of RNA have been investigated using 2-aminopurine (Ballin et al., 2007, 2008). In these studies, extrahelical/bulged, base paired, and looped bases were identified by the fluorescence intensities and lifetimes of selectively substituted 2-aminopurines. These studies are distinguished by their use of control single-stranded sequences to isolate structural from temperature and base context effects on the fluorescence properties of 2-aminopurine. Our studies use these important contributions to understand the structures and thermodynamics of repeated (CAG) sequences.
2. Materials and Methods 2.1. Materials Oligonucleotides from Integrated DNA Technologies (Coralville, IA) were synthesized using standard phosphoramidite chemistry and purified using denaturing gel electrophoresis with 7 M urea and 8% polyacrylamide (Table 7.1). A 5-base standard was used to identify DNA strands based on their length, and the desired band was excised and then electrocuted from a dialysis bag. Following buffer exchange using gel filtration chromatography (NAP-10, GE Healthcare), the DNA was lyophilized and resuspended in buffer. Three-way junctions were formed by annealing equimolar quantities of 50- and 74-base oligonucleotides, with the longer DNA strand containing a central (CAG)8 with flanking 25-base regions that complement the shorter strand (Fig. 7.1). Measurements were conducted in buffers comprised of 10 mM Tris/TrisHþ (pH ¼ 7.9) with 10 mM MgCl2 and 50 mM NaCl or 10 mM H2PO4/HPO42 (pH ¼ 7) with 50 mM NaCl. The results show no buffer dependence. Nomenclature for modified oligonucleotides identifies the position of 2-aminopurine within the primary sequence (Fig. 7.1). Starting from the 50 end, individual replacements of adenine with 2-aminopurine in the isolated (CAG)8 hairpin were made in the first, second, third, fourth, fifth, and sixth repeats, and these are designated by 1-APHP, 2-APHP, 3-APHP, 4-APHP, 5-APHP, and 6-APHP, respectively. For the three-way junction, five substitutions notated as 1-APJ, 3-APJ, 4-APJ, 5-APJ, and 6-APJ were made in the first, third, fourth, fifth, and sixth repeats of the central (CAG)8, respectively. Modifications labeled a-APJ and b-APJ were also made in the two (CAG) repeats in the duplex region that precedes the junction.
2.2. Conformational integrity To evaluate how 2-aminopurine influences the secondary structure of repeated sequences, circular dichroism, thermal denaturation, gel electrophoresis, and enzyme recognition are used. In the ultraviolet spectral
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Table 7.1 Single-stranded oligonucleotides
a b c d e
Sequence
Lengtha eb
50 -CACCATGCCGGTA TTTAAA CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG TACGTA CTGCAGCTCGAGG-30 0 5 -CACCATGCC GGT ATT TAA ACAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG TACGTA CTGCAGCTCGAGG-30 c 50 -CCTCGAGCTGCAG TACGTA CTGCTG─CTGCTG TTTAAA TACCGGCATGGTG-30 d 0 5 -CACCATGCCGGTA TTTAAA CAGCAG CAGCAG TACGTA CT GCAGCTCGAGG-30 0 5 -CAG CAG CAG CAG CAG CAG CAG CAG-30 50 -CAG CAG CAG CAG CAG CAG CAG CAG-30 e 50 -G AAA CAG CAG TTTT CTG CTG TTT C-30 (DS-CAG) 50 -AAA CAG CAG-30 (SSb-CAG) 50 -CAG CAG CAG-30 (SS-CAG)
74
720,500
74
708,700
50
462,500
50
484,500
24 24 24
237,300 225,500 210,900
9 9
86,300 78,000
Length in bases. Extinction coefficients (M 1 cm 1) of the unfolded single strands. Seven variants of this oligonucleotide were used, each with a single substitution of 2-aminopurine, indicated by the underlined bases. Connecting line represents the region corresponding to the (CAG)8. Six different substitutions of 2-aminopurine were made in (CAG)8.
region, base–base interactions dominate the circular dichroism response to provide a sensitive indication of nucleic acid structure (Cantor and Schimmel, 1980). The hairpins adopted by (CAG) repeats have a conformation that is most similar to B-form DNA, which suggests that canonical G/C base pairs drive folding (Paiva and Sheardy, 2004). With respect to the unmodified sequences, similar circular dichroism spectra indicate that single 2-aminopurines throughout (CAG)8 have a minimal impact on conformation (Fig. 7.2). To gain an energetic perspective, melting temperatures, enthalpy changes, and entropy changes associated with global denaturation were derived from absorbance changes at 260 nm, and similar thermodynamic values with and without 2-aminopurine indicate that the stabilities of repeat structures are not perturbed by modification (Tables 7.2 and 7.3). To monitor DNA shape, nondenaturing gel electrophoresis shows that the mobility of (CAG)8, 24-base oligonucleotide is comparable to that of a 12 base pair duplex, and no changes with 2-aminopurine were observed (Degtyareva et al., 2009). For the three-way junction, only one band with a mobility that is distinct from its constituent single strands was observed (Degtyareva et al., 2010). Using restriction digestion at the two sites adjacent
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A
B
C
Figure 7.1 (A) Structure for the hairpin formed by (CAG)8. (B) Structure for the three-way junction formed with (CAG)8. Restriction enzyme sites for DraI and BsaAI are indicated in bold and underlined. Below this structure is the duplex that comprises the three-way junction. The connecting lines represent the position of the abstracted repeat sequence. (C) Single- and double-stranded oligonucleotides used as structural references. In all these oligonucleotides, the positions of 2-aminopurine substitution are underlined and/or enumerated.
to the strand junction, gel electrophoresis shows proper strand alignment by liberation of the terminal 16 base pair fragments. Again, 2-aminopurine does not influence these results.
2.3. Calculation of thermodynamic parameters Because thermal denaturation produces a common single-stranded state, differences in the thermodynamic parameters between the variants can be attributed to the low-temperature folded forms. Changes in the extinction coefficients of the bases and the fluorescence quantum yield of 2-aminopurine permit the folded and unfolded states to be defined, from which the energetic factors that impact global and local structure are deduced.
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(CAG)8 1-APHP
20
6-APHP 5-APHP 4-APHP
Ellipticity (m deg)
10
3-APHP 2-APHP
0
–10
–20 200
250
300 Wavelength (nm)
350
400
Figure 7.2 Circular dichroism spectra of unmodified and 2-aminopurine modified (CAG)8 sequences. The similarity in the spectra suggest that 2-aminopurine has no significant effect on the conformation of (CAG)8. Spectral shapes are consistent with a B-like duplex stem (Paiva and Sheardy, 2004).
Standard methods of extracting thermodynamic information are used to generate plots of fraction of folded species, y, versus temperature (Mergny and Lacroix, 2003). Different baselines corresponding to the low- and hightemperature states are used to estimate the uncertainties in the derived quantities. Melting temperatures are determined as the intersection point between the y dependence on temperature and the median between the two baselines. A more thorough thermodynamic analysis uses a two-state analysis to extract enthalpy and entropy changes after accounting for the molecularity of the transitions. Data was analyzed between y ¼ 0.2–0.8, where intermediate temperatures give relatively comparable concentrations of both folded and unfolded forms. Changes in entropy and enthalpy were measured for triplicate samples.
2.4. Oligonucleotide concentration Structural references are used to deduce the secondary structures of repeated sequences, and such comparisons must consider oligonucleotide concentrations. A sensitive method utilizes the high fluorescence quantum yield of unconjugated 2-aminopurine, and the exonuclease BAL-31 cleaves the fluorogenic nucleotide from the oligonucleotide. Concomitant base unstacking in dilute solution results is a significant enhancement in
Table 7.2 Thermodynamic data for (CAG)8 hairpin
a b c d
Modification
Tm ( C)a
DH (kcal/mol)a
DS (cal/mol/K)a
Tm ( C)b
DH (kcal/mol)b
DS (cal/mol/K)b
Kq (M 1)
1-APHP 2-APHP 3-APHP 4-APHP 5-APHP 6-APHP Unmodified SS-CAG DS-CAG
58.8 (1.0)d 60.1 (1.4) 60.3 (1.0) 61.1 (1.0) 59.2 (1.4) 60.2 (1.0) 57.4 (0.6)
32.0 (1.9) 28.5 (2.4) 29.9 (4.7) 32.1 (1.4) 28.9 (1.5) 29.5 (3.7) 29.0 (1.5)
96 (6) 86 (8) 90 (14) 96 (4) 87 (5) 88 (11) 88 (5)
NDc 52.8 (0.3) 54.4 (0.9) 50.4 (0.6) 47.0 (1.0) 51.8 (0.3)
ND 31.2 (0.9) 32.7 (1.3) 32.9 (2.9) 20.0 (2.0) 32.7 (1.3)
ND 96 (3) 100 (4) 95 (9) 61 (6) 100 (4)
9.8 (0.5) 5.0 (0.5) 9.1 (0.8) 11.8 (1.0) 14.8 (1.2) 10.6 (0.4)
Data derived from absorbance changes at 260 nm. Data derived from fluorescence changes of 2-aminopurine modified forms of (CAG)8. Values not determined. Standard deviations in parentheses derived from three measurements.
9.6 (1.0) 8.2 (0.6)
Table 7.3 Thermodynamic data for (CAG)8 three-way junction
a b c d
Modification
Tm ( C)a
DH (kcal/mol)a
DS (cal/mol/K)a
Tm ( C)b
DH (kcal/mol)b
DS (cal/mol/K)b
Kq (M 1)
b-APJ a-APJ 1-APJ 3-APJ 4-APJ 5-APJ 6-APJ Unmodified Duplex
75.9 (0.4)d 77.2 (0.1) 76.6 (0.1) 76.8 (0.8) 76.4 (0.1) 75.9 (0.3) 76.4 (0.6) 77.2 (0.2) 81.4 (0.3)
220 (22) 199 (20) NDc ND 194 (19) ND 217 (22) 199 (20) 243 (25)
606 (61) 543 (54) ND ND 522 (53) ND 600 (60) 540 (54) 665 (67)
77.0 (0.6) 78.7 (0.3) ND 77.0 (1.7) 77.1 (0.6) 77.6 (1.6) 76.5 (0.3)
238 (24) 237 (32) ND 224 (22) 190 (20) ND 250 (25)
630 (63) 650 (65) ND 606 (61) 516 (52) ND 685 (69)
7.5 (0.2) 9.8 (0.5) 11.1 (0.2) 15.0 (0.2) 13.5 (0.7) 15.2 (0.7) 14.9 (0.4)
Data derived from absorbance changes at 260 nm. Data derived from fluorescence changes of 2-aminopurine modified forms of the three-way junctions. Values not determined. Standard deviations in parentheses derived from three measurements.
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fluorescence intensity, which is then related to concentration using a standard curve. Typically, 5–20 nmol of DNA (in base pairs) were mixed with 0.5 units of BAL-31 in digestion buffer (20 mM Tris–HCl, pH 8.0, 600 mM NaCl, 12 mM CaCl2, 12 mM MgCl2, 1 mM EDTA) in a total volume of 50 mL and incubated at 30 C for 20–30 min. The reaction mixture was then diluted with buffer, and fluorescence intensities of the standard and digested samples were measured. Alternatively, because the sequences of the oligonucleotides are known, concentrations were also calculated using extinction coefficients for the cleaved nucleotides (Cavaluzzi and Borer, 2004). Finally, concentrations of intact oligonucleotides were determined using extinction coefficients at 260 nm derived from the nearest-neighbor approximation, with the small contribution from 2-aminopurine (1000 M 1 cm 1 at 260 nm) included in the calculation (Fox et al., 1958). To account for the effect of temperature, high-temperature posttransition baselines were extrapolated back to 25 C. Variations in DNA concentration determined by all three methods were less than 10%.
2.5. Structural references To relate the fluorescence from the 2-aminopurine labeled repeated sequences to particular secondary structural elements of DNA, single- and doublestranded references are used (Ballin et al., 2007, 2008) (Fig. 7.1). Nine-base oligonucleotides provide the environments for 2-aminopurine in solventexposed, single-stranded DNA, and a 2-aminopurine/thymine base pair in a duplex stem provides a reference for the fluorophore in a base-stacked and solvent-sequestered state. Although structurally analogous to the adenine/ thymine pair, the 2-aminopurine/thymine pair has faster proton exchange rates with the solvent and lower thermal stability, indicative of less effective stacking within the duplex (Lycksell et al., 1987; Patel et al., 1992; Xu et al., 1994). In both reference oligonucleotides, the immediate base environment of the 2-aminopurine is identical to that in the repeated sequence. In addition to secondary structure and neighboring bases, temperature also influences the fluorescence quantum yield of 2-aminopurine (Fig. 7.3). To dissect these contributions during thermal denaturation, the ratios of fluorescence intensities from the single-stranded reference and repeated sequences are used, as the former maintain an unfolded state over the entire temperature range. At high temperatures, similar fluorescence intensities indicate that both types of DNA achieve the same unfolded state (Figs. 7.3 and 7.4).
2.6. Acrylamide quenching To complement information derived from the inherent fluorescence of 2-aminopurine, extrinsic fluorescence quenching by acrylamide is also used to assess solvent exposure (Lakowicz, 2006). By relating quenching
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Figure 7.3 Emission intensities of 3-APHP (dashed) and SS-CAG (solid) as a function of temperature. The intensity change associated with the single-stranded reference is due solely to temperature, as this oligonucleotide is unfolded over the entire temperature range. The inset shows the normalized intensity for 3-APHP using the reference to account for temperature effects on the emission intensity. Because of denaturation of the (CAG)8 hairpin at high temperature, both oligonucleotides achieve comparable intensities.
efficiency to secondary structural elements of DNA, structural variations within the oligonucleotides are inferred (Ballin et al., 2007, 2008). Standard Stern–Volmer analysis is used to extract quenching constants, Kq, for the 2-aminopurines (Fig. 7.5, Tables 7.2 and 7.3). The smallest quenching constants are associated with bases that are most protected from the solvent, as in a duplex. As the fluorogenic base becomes more exposed to the aqueous solution, as for single-stranded DNA, the quenching constant increases.
3. Structure and Thermodynamics of Isolated and Integrated (CAG)8 3.1. (CAG)8 hairpin The overall conclusion from 2-aminopurine studies of (CAG)8 alone is that this repeated sequence favors a hairpin displaying suppressed fluorescence in the stem and enhanced fluorescence in the loop (Figs. 7.1 and 7.4). Folding driven by strand association is indicated by the similar behaviors of the distant modifications 2-APHP/3-APHP and 6-APHP. These three variants exhibit relatively lower emission intensities when compared to single-stranded
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Figure 7.4 (Top) Emission intensities of 2-aminopurine-modified (CAG)8 as a function of temperature normalized using the emission from SS-CAG (lex ¼ 307 nm, lem ¼ 370 nm). (Bottom) Normalized emission intensities of the modified three-way junctions as a function of temperature. To the right of each graph are the models for the secondary structure based on fluorescence intensities. The graphic emphasis at each position indicates its level of solvent exposure.
DNA, and these intensities are comparable at high temperature. These two observation support base stacking in the folded, low-temperature form of (CAG)8 and are consistent with the propensity of repeated sequences to form into hairpins with B-like conformations in the stem (Paiva and Sheardy, 2004). Incorporation of the resulting purine–purine mismatches in the stem of (CAG)8 is gauged using a duplex with a 2-aminopurine/thymine base pair. Comparable intensities for matched and mismatched pairs involving 2-aminopurine indicate their comparable degrees of stacking with neighboring bases, a conclusion that is supported by NMR studies of duplexes containing adenine/adenine mismatches (Mariappan et al., 1998). The other major structural element identified by 2-aminopurine is a loop between the duplex. Relative to the single-stranded reference, 5-APHP has a higher
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Figure 7.5 Stern–Volmer plots of the acrylamide quenching of 2-aminopurine variants of (CAG)8 (top) and the three-way junction (bottom). F0 and F are the fluorescence intensities in the absence and presence of acrylamide, respectively. Linear fits were used to derive quenching constants using the standard Stern–Volmer model.
intensity that diminishes with temperature. This is consistent with a constrained loop that relaxes to enhance base stacking and the associated solvent sequestration in the single-stranded form. An important advantage of
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2-aminopurine is the strong effect of base stacking on fluorescence quantum yield, thereby resolving finer details of secondary structure. For (CAG)8, transitional behavior between the stem and loop motifs is evident. For 1APHP, its intensity is comparable to the single-stranded reference over the entire temperature range, consistent with frayed, unpaired terminal repeats. Features of both the stem and loop are exhibited by 4-APHP, which has an intermediate fluorescence intensity and smaller fluorescence enhancement. This behavior is considered in the context of models of repeat folding, which indicate that stacking interactions in the stem are maximized by the tetraloop AGCA (Fig. 7.1; Hartenstine et al., 2000). In (CAG)8, these bases comprise the fourth and fifth repeats, yet variations in the fluorescence properties of 2-aminopurine substitutions in these repeats suggest an asymmetric loop environment. Local variations in the interactions with neighboring bases could be dictating the structure of the loop (Mariappan et al., 1996; Senior et al., 1988; Varani, 1995). Structural patterns deduced from fluorescence are supported by acrylamide quenching studies, which show clustering into three groups (Fig. 7.5, Table 7.2). The least efficient quenching is observed for 2-APHP and the duplex reference, which supports protection for 2-aminopurine within a duplex stem from this external quenching agent. Higher solvent exposure comparable to the single-stranded reference is observed for 1-APHP, 3-APHP, 4-APHP, and 6-APHP. While the frayed ends of 1-APHP and the single-stranded reference are expected to be structurally similar, the fluorescence properties of 3-APHP, 4-APHP, and 6-APHP indicate that these 2-aminopurine substitutions are more solvent sequestered. Thus, these similarities suggest that acrylamide quenching constants should serve as general guides to the structures of repeated sequences. The largest quenching constant is observed for 5-APHP, consistent with the high fluorescence intensity from this region of the loop. Quenching is as efficient as observed for 2-aminopurine at the exposed bulge of a RNA duplex, but is smaller than that of the free nucleobase (Ballin et al., 2007; Degtyareva et al., 2009). As utilized in structural studies, denaturation of (CAG)8 produces a single-stranded oligonucleotide, so high temperature fluorescence spectra provide an important structural reference. Furthermore, temperaturedependent fluorescence changes allow the fractional conversion from folded to unfolded states to be derived, from which enthalpy and entropy changes are determined using Arrhenius analysis (Table 7.2). A context for interpreting local energetic changes is provided by global unfolding using hyperchromic absorbance changes at 260 nm. These enthalpy and entropy changes are similar to values obtained from calorimetric analysis, which suggests that neglecting heat capacity changes does not significantly compromise interpretation of the results (Amrane et al., 2005; Paiva and Sheardy, 2004). For 2-APHP, 3-APHP, and 6-APHP, local enthalpy and entropy changes derived from fluorescence are similar to the global values.
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This consistency suggests that base pairing and stacking the stem dictate the overall stability of the folded hairpin. In contrast, 5-APHP has both smaller enthalpy and entropy changes for denaturation relative to the global values. These energetic features complement the relatively high fluorescence from this region of the hairpin and support limited base interactions in the loop of the hairpin. Thermodynamic measurements also provide insight into how the mismatch influences the stability of the stem. Global melting temperatures derived from absorbance are 6–8 C higher than those derived from fluorescence. This behavior suggests that neighboring base interactions with 2-aminopurine weaken and thereby enhance solvent exposure prior to global denaturation (Lycksell et al., 1987; Nordlund et al., 1993).
3.2. (CAG)8 three-way junction Fluorescence from 2-aminopurine shows that integration of (CAG)8 into a duplex disrupts intrastrand association within the repeat sequence to produce an open and solvent-exposed loop (Figs. 7.1 and 7.4). To understand this context-dependent structural change for (CAG)8, seven modifications were used to map solvent exposure throughout the three-way junction. Within the duplex arms, (CAG) repeats base pair with opposing (GTC) repeats in the complementary strands, as shown by b-APJ and a-APJ. Emission intensities from these variants are lower than the single-stranded reference and increase with denaturation at high temperature, and both observations support base stacking of 2-aminopurine that is relieved with thermal denaturation. However, the strand junction perturbs base pairing and stacking within the duplex, as indicated by the higher intensities of b-APJ and a-APJ relative to the duplex reference with a 2-aminopurine/ thymine base pair. Furthermore, a-APJ has a higher intensity than b-APJ, suggesting that the level of solvent exposure increases with proximity to the junction. This trend in solvent exposure continues into the repeated (CAG)8 sequence, with 1-APJ acting as single-stranded DNA over the entire temperature range. Collectively, the fluorescence properties of these three modifications indicate that the transition from duplex to repeated sequence is accompanied by disrupted base interactions that increase solvent exposure of the bases. This disturbance appears to be transmitted into the repeated sequence, with 3-APJ, 4-APJ, 5-APJ, and 6-APJ exhibiting similarly high fluorescence intensities when compared with the single-stranded reference. In addition, mirroring the loop region of the (CAG)8 hairpin, these four variants of the three-way junction display solvent exposure that diminishes at higher temperatures, suggesting that constraints induced by the junction relax when the three-way junction denatures. This openness of the repeated sequence strikingly contrasts with the folded (CAG)8, as most clearly demonstrated by the third and sixth repeats. Hairpin formation in (CAG)8 alone is shown by comparable
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and relatively low levels of solvent exposure in 3-APHP and 6-APHP. In contrast, similarly placed 2-aminopurines in 3-APJ and 6-APJ are more solvent exposed when compared with the single-stranded reference. Acrylamide quenching studies support a model in which solvent exposure becomes comparable to single-stranded DNA in the vicinity of the strand junction and is consistently high in the core of the repeated sequence (Fig. 7.5, Table 7.3). Quenching for b-APJ and the duplex reference are comparable, which suggests that the b modification is protected from extrinsic quenching by base stacking and pairing. Quenching becomes increasingly efficient proceeding toward the junction from a-APJ and onto 1-APJ. Quenching constants that are consistently higher than singlestranded DNA are observed for 3-APJ, 4-APJ, 5-APJ, and 6-APJ. Additional support for the open conformation throughout the integrated (CAG)8 is provided by the similarity of these quenching constants when compared with 5-APHP in the loop of the (CAG)8 hairpin. To understand the thermodynamic factors that contribute to the stability of the three-way junction, thermal denaturation was followed using absorbance and fluorescence spectral changes (Table 7.3). After dissecting the repeated sequence, the resulting duplex is shown to dominate the stability of the three-way junction. Specifically, enthalpy and entropy changes for the three-way junction are slightly ( 20%) lower than the values for the duplex. Furthermore, the melting profile of the three-way junction is monophasic with no evidence of a transition associated with the (CAG)8 hairpin. Local unfolding was monitored using the fluorescent variants, and the similarities of the melting temperatures, enthalpy changes, and entropy changes relative to the values derived from absorbance indicate that local structural changes track global unfolding. Collectively, these observations suggest that the stability of the three-way junction is dictated by its duplex arms with slight instability induced by the repeated sequence. The overall conclusion is that DNA context critically impacts the structure of (CAG)8. Enzymatic probes also highlight the distinctive features of (CAG) repeats when compared with (CTG) repeats (Pearson et al., 2002). In slipped intermediates, enzymatic recognition of the latter repeats is consistent with their folded, hairpin structures that also form in the analogous isolated repeats. In contrast, (CAG) repeats preferentially interact with single-strand specific enzymes and proteins, indicative of their random coil conformation. Such a conformation differs from the folded and largely protected hairpins favored by isolated (CAG) hairpins. This behavior again supports the dominant effect of the duplex arms and the strand junction on the structure of such repeated sequences. The significance of these earlier results and our studies using 2-aminopurine lies in the importance of secondary structure in the biological recognition and processing of repeated sequences via enzyme recognition (Kovtun and McMurray, 2008; Pearson et al., 1997).
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4. Conclusions The adenine isomer 2-aminopurine provides an avenue to understand the secondary structures of repeated DNA sequences, and a profound difference is noted when (CAG)8 is isolated from versus integrated into a duplex of canonical base pairs. Alone, (CAG)8 forms a hairpin in which folding is driven by duplex formation in the stem. In contrast, such distant intrastrand interactions are absent when (CAG)8 is incorporated into a duplex. This structural distinction suggests that the duplex has a significant effect on the structures of repeated sequences. These results provide the foundation for understanding the secondary structures favored by larger repeated sequences that are implicated in debilitating neurological diseases. Such structures will be elucidated from the repeat level resolution of structural and energetic information that is gleaned from 2-aminopurine. By identifying key structural elements favored by repeated sequences, the path to understanding the mechanisms of repeat expansion will have a stronger molecular basis.
ACKNOWLEDGMENTS We are grateful to the National Science Foundation (CHE-0718588) and the Henry Dreyfus Teacher–Scholar Awards Program for the primary support for this work. We also greatly appreciate partial support from the National Science Foundation (CBET-0853692) and the National Institutes of Health (R15GM071370 and P20 RR-016461 (from the National Center for Research Resource)).
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