Investigating the Thermodynamics of UNCG Tetraloops Using Infrared

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Investigating the Thermodynamics of UNCG Tetraloops Using Infrared Spectroscopy Aaron L Stancik, and Eric B. Brauns J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp408496z • Publication Date (Web): 30 Sep 2013 Downloaded from http://pubs.acs.org on October 7, 2013

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The Journal of Physical Chemistry

Investigating the Thermodynamics of UNCG Tetraloops Using Infrared Spectroscopy Aaron L. Stancik† and Eric B. Brauns* Department of Chemistry, University of Idaho, Moscow, Idaho, 83844-2343 *Corresponding author: [email protected] †Present address: Anatek Labs, Inc. Moscow, Idaho, 83843

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ABSTRACT. Using infrared (IR) absorption spectroscopy, we have explored the folding thermodynamics of the UNCG class of RNA hairpin tetraloops (N = U, A, C, or G). Without the need to introduce non-native probes, IR spectroscopy makes it possible to distinguish specific structural elements such as base pairing vs. base stacking or loop vs. stem motions. Our results show that different structural components exhibit different thermodynamics. Specifically, we have found that tetraloop melting proceeds in a thermally sequential fashion where base pairing in the stem is disrupted before (i.e., at a lower temperature) base stacking along the entire chain. In addition, for N = A, our data argue that the structure immediately surrounding the adenine is particularly stable and melts at a higher temperature than either base pairing or base stacking interactions. Taken together, these results suggest that hairpin loop formation is not a simple two-state process, even in the equilibrium limit. KEYWORDS RNA, hairpin loop, melting, folding, IR


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INTRODUCTION The current paradigm in our understanding of RNA folding is that it is hierarchical—larger, more complex tertiary structures are built from pre-formed, independently stable secondary structures.1,2 Of the different types of secondary structural motifs found in all RNA molecules, hairpin loops are by far the most ubiquitous.3 A hairpin loop forms when a section of RNA folds back on itself creating a loop of unpaired bases and a helical stem comprised of base pairs. While loops of many sizes exist in nature, tetraloops (i.e., four unpaired bases in the loop) are the most common. Moreover, of the 256 possible tetraloop sequences, UNCG (N = U, A, C, or G) occurs more often than any other. One reason why the UNCG tetraloops are so pervasive is that they are exceptionally stable and function as nucleation sites for the formation of more complex architectures.4 Despite the central role that hairpin loops play in RNA folding, we have just begun to study them in detail. In contrast to their deceptively simple structure, recent time-resolved experiments suggest that the folding mechanism of tetraloops is surprisingly complex.5,6 These experiments show that tetraloop folding proceeds through a series of intermediate states rather than via a simple two-state mechanism. Using infrared spectroscopy, our work dissects the mechanism further by monitoring the kinetics at different wavenumbers that correspond to specific structural moieties in the RNA. For example, in our earlier time-resolved study of UCCG tetraloop folding, the observed kinetic behavior was wavenumber dependent.6 From these data, we argued that the first step in UCCG tetraloop folding was the formation of base stacking interactions (native and non-native) along the RNA strand. Using IR spectroscopy to probe RNA folding has two distinct advantages. (1) The intrinsic vibrational transitions of the RNA can be probed directly so that non-native probes, which have 3 ACS Paragon Plus Environment


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the potential to alter the RNA’s structure, are not required. (2) The vibrational transitions are sensitive to specific molecular groups within an RNA molecule.7 This allows one to glean much more specific structural information from the data. For example, IR spectroscopy can distinguish between AU and GC base pairing interactions as well as between base stacking and base pairing interactions.6,8 It has even been used to determine the base composition of an RNA molecule.9 In this paper, we use IR spectroscopy to explore the thermodynamic properties of all four UNCG tetraloops. Although this is a preliminary step to performing time-resolved experiments, the results have provided new insight. Just as we’ve seen wavenumber dependent kinetics using IR spectroscopy, certain sequences exhibit wavenumber dependent thermodynamics. Observing this behavior in the equilibrium limit greatly improves our understanding of tetraloop structures and underscores the view that they are much more complex than once thought. EXPERIMENTAL METHODS In nature, most UNCG tetraloops are closed by GC base pairs.10,11 In addition, a stem comprised of at least two base pairs is required in order to form a stable hairpin.12 For these reasons, the RNA oligonucleotides used in this work have the generic sequence, 5′-gcUNCGgc3′ (N = U, A, C, or G). The bases written in uppercase are the unpaired bases found in the loop and those written in lowercase comprise the stem. The HPLC purified oligonucleotides were purchased from Integrated DNA Technologies. Samples were prepared by dialyzing against 100 mM phosphate buffer (pH = 7.2) and then lyophilizing against D2O three times to remove labile protons. The final solutions contained RNA at a nucleotide concentration of ~16 mM. Our own dilution experiments as well as previously published work verify that this concentration is below the level needed for duplex formation.4,13


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Samples were placed in custom built cells comprised of two CaF2 windows separated by a Teflon spacer that defines the cell path length. The spacer also divides the cell into two compartments; one for the sample (RNA and buffer) and one for the reference (buffer alone). In this way, sample and reference measurements are obtained by simply translating the cell from side to side. For the experiments described here, the cell path length was 53 µm. The relative path lengths of the sample and reference compartments were determined by measuring interference fringes and were found to be within 0.3 µm of each other. The CaF2 windows are then placed in a copper housing which is then mounted on a larger copper block that is coupled to a circulating water bath for temperature control. The cell temperature is maintained to within ±0.1°C. Temperature dependent IR absorption spectra were recorded on a MIR 8025 FTIR spectrometer (Newport). The cell assembly was mounted to a computer controlled translation stage and placed in a custom built sample compartment. The stage allowed sample and reference spectra to be recorded without compromising the sample compartment purge. Temperature dependent spectra were recorded in 2°C increments from 20°C to 90°C. At each temperature, 512 co-added scans were recorded for the sample and reference. Rather than recording 512 scans for the reference followed by 512 sample scans, the cell was translated back and forth so that sample and reference spectra were recorded alternately. This was done to minimize any longterm baseline drift. All experiments were repeated three times to ensure reproducibility. The melt curves in Figure 2 were fit to a model described in the Results section. In cases where it was determined that a fitting parameter was the same for data at different wavenumbers, the data were re-fit using a global fitting algorithm. Using global analysis, multiple datasets are fit simultaneously while sharing the parameters that were previously determined to be common 5 ACS Paragon Plus Environment


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to them. This improves the reliability of the fitting procedure by reducing the number of parameters. In addition, it is particularly beneficial when comparisons are to be made between related datasets. RESULTS Temperature dependent IR spectra for each of the UNCG hairpin loops are shown in Figure 1. The sensitivity of IR spectroscopy to RNA hairpin structure is immediately evident. When unfolded, there are no secondary structural contacts and the IR spectrum of an RNA molecule is essentially the weighted sum of the IR spectra for each of the individual monomers comprising the sequence.9 The sequences used in this work differ by only a single nucleotide base; accordingly, their unfolded spectra (high temperature; red lines) are very similar. As the temperature is lowered, the RNA begins to fold and the local environments surrounding each base changes. This alters their vibrational characteristics and manifest as changes in their IR spectra. As a result, the spectrum of a folded RNA molecule contains additional structural information. For the sequences discussed in this work, the spectra of the folded oligomers (low temperature; blue lines) are dissimilar, suggesting that—despite their sequence similarities— their structures are very different.


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Figure 1. Temperature dependent IR absorption spectra (lower plots) and corresponding difference spectra (upper plots). The sequence corresponding to each dataset is indicated in the upper right hand corner. The vertical lines indicate the wavenumbers where the thermodynamics were evaluated. In all, the blue lines indicate the lowest temperature spectrum, the red lines indicate the highest temperature spectrum, and the light gray lines indicate spectra at intermediate temperatures. Difference spectra were calculated by subtracting the lowest temperature spectrum from each higher temperature spectra.



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The sensitivity of IR spectroscopy to RNA conformational changes is highlighted in the difference spectra. These are shown in the upper plots of Figure 1. Difference spectra are obtained for each oligomer by subtracting the lowest temperature spectrum from each higher temperature spectra. Viewing the data in this manner accentuates the spectral changes that occur as the RNA melts. The spectral assignments that follow are taken from the same reference.7 In all the sequences studied, there is a prominent difference feature at ~1574 cm-1. This is due to a C=N ring vibration of guanine and primarily reports on base stacking interactions. Also present in all samples is a positive difference feature at ~1665 cm-1 accompanied by a simultaneous negative feature at ~1686 cm-1. These are due to the combined effects of a guanine C6=O6 stretch and a cytosine C2=O2 stretch that occur when a GC base pair is broken. Since GC base pairs are found only in the stem of our samples, absorption changes at ~1665 cm-1 or ~1686 cm-1 serve as specific indicators of loop closure. Unique among the hairpins studied is the UACG tetraloop. As the only oligomer that contains an adenine, it has a positive difference feature at 1618 cm-1 that is due to an adenine C=N stretch. This transition is very useful since it is “loop specific”. In all the other oligomers, the base substituted in the N position of the loop is already present elsewhere in the sequence. However, in the UACG tetraloop, the adenine in the loop is the only one present in the sequence. As a result, any observed spectral changes are due to structural changes affecting the ring vibrations on this base within the loop. This localized “probe” can help distinguish between structural changes in the stem from those in the loop. Before continuing, we should note that the spectral assignments just discussed are by no means absolute. For example, changes at ~1574 cm‐1 was described as an indicator of base stacking interactions. This does not mean that it reports on base stacking only and is completely 8 ACS Paragon Plus Environment

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unaffected by base pairing. Because the molecular groups are connected by C–C bonds, when a guanine forms a base pair, the change in the dipole moment of the C=O group will be felt by the ring vibrations. As a result, the changes at ~1574 cm‐1 could still change even if no stacking is occurring. However, changes at this wavenumber are mostly sensitive to stacking interactions and less sensitive to base pairing. The same is true for the spectral features previously attributed to base pairing. As a final comment, it is important to keep in mind that RNA folding is a concerted process and that base stacking and base pairing interactions do not occur independently of each other. Despite this, it remains useful to separate them for the purposes of discussion. Thermodynamic information is obtained from the spectral data by first plotting melt curves (absorbance vs. temperature) for key wavenumbers that are sensitive to conformational changes. Normalized melt curves along with fits to a thermodynamic model (vide infra) for the four oligomers studied here are shown in Figure 2. Because the IR spectra at each temperature are recorded at thermal equilibrium, it is possible to extract thermodynamic information from the melting data. To do so, the melt curves are fit to a phenomenological two‐state model14,15 A (T ) = a − ( a − b ) fU

(1)

where fU is the fraction of the molecules that are unfolded, a is the absorbance of the unfolded molecules, and b is the absorbance of the folded molecules. In a two-state model, fU is related to the equilibrium constant Keq by

fU =

K eq 1 + K eq

(2)



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The equilibrium constant is then related to the Gibbs free energy ∆G according to:

K eq = exp ( −∆G RT )

(3)

Using the Gibbs-Helmholtz equation, the temperature dependence of the Gibbs free energy is expressed in terms of the enthalpy of unfolding (∆H), the melting temperature (Tm), and the change in heat capacity (∆CP):

T   T   ∆G (T ) = ∆H (T ) 1 −  + ∆CP  T − Tm − T ln  Tm   Tm  

(4)

For this work, we set ∆CP equal to zero to reduce the number of parameters in the thermodynamic model. Although this sacrifices some accuracy, it increases the reliability of the fit substantially, and the same general conclusions can still be drawn. Once the fit parameters are obtained, eq 1 can be rearranged to convert the raw data and the fit functions from absorbance to fraction unfolded. This effectively normalizes the melt curves (since 0 ≤ fU ≤ 1) and facilitates visual comparison between different samples as shown in Figure 2.


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Figure 2. Melting curves at specific wavenumbers for each of the four sequences (indicated in the upper left hand corner of each plot). The solid lines through the data points are fits to eq 1. For N = C, the melt curves were identical at both wavenumbers and only a single fit was obtained (solid black line). DISCUSSION Taken individually, each melt curve in Figure 2 is not particularly remarkable. Each is sigmoidal, which is a defining feature of a simple two-state transition, and the thermodynamic parameters (summarized in Table 1) are in good agreement with known values.12,13,16,17 However, using IR spectroscopy, we have simultaneously probed multiple vibrational transitions within a single sample; each of which corresponds to different structural features. In effect, we are able to monitor a single event (i.e., unfolding) from different “perspectives”. The results are striking; the melt curves recorded at different wavenumbers do not overlap for the N = U, A, and 11 ACS Paragon Plus Environment


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G oligomers. This means that the observed thermodynamics, specifically the melting temperatures, are wavenumber dependent.

Table 1. Thermodynamic parameters N

∆Ha (kJ mol-1)

Tm (°C)b 1574 cm-1

Tm (°C)b 1618 cm-1

Tm (°C)b 1665 cm-1

U

113.0

54.5

-

51.3

A

116.3

50.8

52.5

47.9

C

104.6

57.3

-

57.3

G

187.0

61.9

-

59.9

-1

a. errors less than ± 6 kJ mol ; b. errors less than ± 0.3°C What these data suggest is that, even in the equilibrium limit, the folding of RNA hairpins is far more complex than previously thought. Most notably, the folding is not, strictly speaking, “two‐state”. Since a purely two‐state transition would give the same thermodynamic parameters regardless of the probe wavenumber, the wavenumber dependent thermodynamics is strong evidence of a more involved folding mechanism. What constitutes a complex folding mechanism? Folding complexity is a consequence of two possible phenomena. One is that folding proceeds sequentially along a single path but is interrupted by one or more intermediates (a local minima in the energy landscape perspective). Another is a parallel mechanism where folding proceeds along at least two unique pathways that converge at the same folded state. In a parallel mechanism, each pathway can, in turn, be sequential. Our data seems to indicate a sequential mechanism according to the following argument. Since enthalpy is a state function, its value depends only on the initial and final states of the system (i.e., it is independent of the pathway between the two states). For all the hairpins studied, the enthalpies were wavenumber independent which suggests that the folded and unfolded states


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were the same and therefore connected by a single pathway (note that this is not absolutely conclusive since it is possible for different states to be energetically degenerate). The support for the sequential mechanism lies in the wavenumber dependence of the melting temperatures which argues that different structural features (e.g., base pairs and base stacks) melt at different temperatures. Specifically, the base pairs melt (i.e., are disrupted) at a lower temperature than the base stacks for the N = U, A, and G tetraloops. In other words, as the temperature is increased, base pairs in the stem are disrupted and the loop begins to open. This results in a structure that retains some of its native stacking contacts which remain intact until the temperature is increased further. Furthermore, of the three wavenumbers probed for the UACG tetraloop, the highest melting temperature was observed for the data corresponding to the adenine found only in the loop. This suggests that the bases in the loop adopt a particularly stable configuration. Although our data show that this is true for only one of the oligomers, it is plausible that this could be a general feature of all the UNCG hairpins. CONCLUSIONS Although the motivation for the work presented here was to prepare for time-resolved experiments, the results were surprisingly significant in their own right. In addition to providing the information necessary to carry out meaningful kinetic experiments, this work offers considerable insight into the folding mechanism of UNCG hairpin tetraloops. We have found that tetraloop melting proceeds in a thermally sequential fashion where base pairing in the stem melts before base stacking along the entire chain. In addition, for N = A, our data argue that the structure immediately surrounding the adenine is particularly stable and has a higher melting temperature than either base pairing or base stacking interactions. These results suggest that



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hairpin loop formation is not a simple two-state process, even in the equilibrium limit. Work is currently underway to explore the wavenumber dependent kinetics of these oligomers. REFERENCES

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Brion, P.; Westhof, E. Hierarchy and Dynamics of RNA Folding. Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 113-37.

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Schroeder, R.; Grossberger, R.; Pichler, A.; Waldsich, C. RNA Folding in vivo. Curr. Opin. Struct. Biol. 2002, 12, 296-300.

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Baumruk, V.; Gouyette, C.; Huynh-Dinh, T.; Sun, J.; Ghomi, M. Comparison between Cuug and UUCG Tetraloops: Thermodynamic Stability and Structural Features Analyzed by UV Absorption and Vibrational Spectroscopy. Nucl. Acids Res. 2001, 29, 4089-96.

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Ma, H.; Proctor, D. J.; Kierzek, E.; Kierzek, R.; Bevilacqua, P. C.; Gruebele, M. Exploring the Energy Landscape of a Small RNA Hairpin. J. Am. Chem. Soc. 2006, 128, 1523-30.

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Stancik, A. L.; Brauns, E. B. Rearrangement of Partially Ordered Stacked Conformations Contributes to the Rugged Energy Landscape of a Small RNA Hairpin. Biochemistry 2008, 47, 10834-40.

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Banyay, M.; Sarkar, M.; Gräslund, A. A Library of IR Bands of Nucleic Acids in Solution. Biophys. Chem. 2003, 104, 477-88.

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Brauns, E. B.; Dyer, R. B. Time-Resolved Infrared Spectroscopy of RNA Folding. Biophys. J. 2005, 89, 3523-30. 14 ACS Paragon Plus Environment

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Thomas, G. J., Jr. Determination of the Base Pairing Content of Ribonucleic Acids by Infrared Spectroscopy. Biopolymers 1969, 7, 325-34.

10. Antao, V. P.; Lai, S. Y.; Tinoco, I. A Thermodynamic Study of Unusually Stable RNA and DNA Hairpins. Nucl. Acids Res. 1991, 19, 5901-05. 11. Zhang, W.; Chen, S. J. Exploring the Complex Folding Kinetics of RNA Hairpins: Ii. Effect of Sequence, Length, and Misfolded States. Biophys. J. 2006, 90, 778-87. 12. Molinaro, M.; Tinoco, I. Use of Ultra-Stable UNCG Tetraloop Hairpins to Fold RNA Structures - Thermodynamic and Spectroscopic Applications. Nucl. Acids Res. 1995, 23, 3056-63. 13. Abdelkafi, M.; Leulliot, N.; Baurmruk, V.; Bednárová, L.; Turpin, P. Y.; Namane, A.; Gouyette, C.; Huynh-Dinh, T.; Ghomi, M. Structural Features of the UCCG and UGCG Tetraloops in Very Short Hairpins as Evidenced by Optical Spectroscopy. Biochemistry 1998, 37, 7878-84. 14. Dyer, R. B.; Brauns, E. B. In Methods Enzymol.; Elsevier Academic Press Inc: San Diego, 2009; Vol. 469, p 353-72. 15. Puglisi, J. D.; Tinoco, I., Jr. Absorbance Melting Curves of RNA. Methods Enzymol. 1989, 180, 304. 16. Proctor, D. J.; Ma, H.; Kierzek, E.; Kierzek, R.; Gruebele, M.; Bevilacqua, P. C. Folding Thermodynamics and Kinetics of YNMG RNA Hairpins: Specific Incorporation of 8-



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Bromoguanosine Leads to Stabilization by Enhancement of the Folding Rate. Biochemistry 2004, 43, 14004-14. 17. Sarkar, K.; Meister, K.; Sethi, A.; Gruebele, M. Fast Folding of an RNA Tetraloop on a Rugged Energy Landscape Detected by a Stacking-Sensitive Probe. Biophys. J. 2009, 97, 1418-27. TOC GRAPHIC


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Temperature dependent IR absorption spectra (lower plots) and corresponding difference spectra (upper plots). The sequence corresponding to each dataset is indicated in the upper right hand corner. The vertical lines indicate the wavenumbers where the thermodynamics were evaluated. In all, the blue lines indicate the lowest temperature spectrum, the red lines indicate the highest temperature spectrum, and the light gray lines indicate spectra at intermediate temperatures. Difference spectra were calculated by subtracting the lowest temperature spectrum from each higher temperature spectra. 147x124mm (300 x 300 DPI)

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Melting curves at specific wavenumbers for each of the four sequences (indicated in the upper left hand corner of each plot). The solid lines through the data points are fits to eq 1. For N = C, the melt curves were identical at both wavenumbers and only a single fit was obtained (solid black line). 109x69mm (300 x 300 DPI)


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TOC graphic 53x36mm (300 x 300 DPI)


Investigating the Thermodynamics of UNCG Tetraloops Using Infrared

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