Exploring the effects of temperature and pressure on the structure

and stability of a small RNA hairpin Caroline Schuabb, Salome Pataraia, Melanie Berghaus, and Roland Winter*

TU Dortmund University, Department of Chemistry and Chemical Biology, Physical Chemistry I

– Biophysical Chemistry, D-44221 Dortmund, Germany

* E-mail: roland.winter@tu-dortmund.de

Supplementary Information

Additional FRET data

Figure SI 3. Fluorescence spectra of the

donor Cy3 and the FRET signal of the

sRNAh in the temperature range from 5 to

95 0C.

Figure SI 4. Fluorescence spectra of the

solo Cy5 fluorophore in the temperature

range from 5 to 95 0C.

Figure SI 1. Fluorescence spectra of the

donor Cy3 and the FRET signal of the

sRNAh at 70 0C in the pressure range from

0.1 to 275 MPa.

Figure SI 2. Fluorescence spectra of the

solo Cy5 fluorophore at 70 0C in the pres-

sure range from 0.1 to 275 MPa.

540 560 580 600 620 640 660 680 700

0.0

5.0x104

1.0x105

1.5x105

2.0x105

2.5x105

3.0x105

0.1 MPa

25 MPa

50 MPa

75 MPa

100 MPa

125 MPa

150 MPa

175 MPa

200 MPa

225 MPa

250 MPa

275 MPaInte

nsity /

a.u

.

Wavelength / nm

640 650 660 670 680 690

1.5x105

2.0x105

2.5x105

3.0x105

3.5x105

4.0x105

Inte

nsity /

a.u

.

wavelength / nm

0.1 MPa

25 MPa

50 MPa

75 MPa

100 MPa

125 MPa

150 MPa

175 MPa

200 MPa

225 MPa

250 MPa

275 MPa

640 660 680 700

5.0x103

1.0x104

1.5x104

2.0x104

2.5x104

3.0x104

3.5x104

4.0x104

4.5x104

Inte

nsity / a

.u.

Wavelength / nm

5 oC

15 oC

25 oC

35 oC

45 oC

55 oC

65 oC

75 0C

85 oC

95 oC

540 560 580 600 620 640 660 680 700 720

0.0

5.0x104

1.0x105

1.5x105

2.0x105

2.5x105

5 oC

15 oC

25 o

C

35 oC

45 oC

55 oC

65 oC

75 0C

85 oC

95 0C

Inte

nsity / a

.u.

Wavelength / nm

Small RNA hairpin in-plane base vibrations sensitive to effects of base stacking followed by

FTIR spectroscopy

a)

b)

Figure SI 5. a) Temperature-dependent normalized and second derivative spectra of the small

RNA hairpin in the wavenumber region from 1600 to 1550 cm-1. Second derivative and normalized

spectra of 5 mg mL-1 small RNA hairpin in 50 mM Tris-HCl buffer + 0.1 mM EDTA from 10 to

95 oC. In the normalized spectra, the IR band at 1590 to 1575 cm-1, related to ring vibrations of C4

= C5 and C5 – C6 of free and base paired guanine, increases with temperature (indicated by the

arrow). The IR band at 1568 to 1564 cm-1, related to ring vibrations of C6 = O6, C5 – C6 and C4

= C5 of free and base paired guanine, decreases with temperature (indicated by the arrow). The

most important bands are indicated by the grey rectangle in the second derivative spectra. The

highlighted bands are: 1576 cm-1, 1564 - 60 cm-1. b) Temperature-dependent changes of the

sRNAh's ring vibrations of free and base paired guanine from 1575 to 1573 cm-1 followed by FTIR

from 10 to 95 oC.

Figure SI 5 shows the temperature-dependent FTIR spectra of the sRNAh in the wavenumber re-

gion from 1600 to 1550 cm-1. In this region of the RNA spectra, two IR bands can be observed.

One IR band around 1590 to 1575 cm-1 and another IR band around 1568 - 1564 cm-1. The latter

band arises from ring vibrations of C6 = O6, C5 – C6 and C4 = C5 of free and base paired guanine.

The former band arises from ring vibrations of C4 = C5 and C5 – C6 of free and base paired

guanine.[1] The intensity increase of the IR band at 1575 cm-1 follows the temperature induced

denaturation of the sRNAh. Whereas the IR band at 1560 cm-1 decreases in intensity, a concomitant

increase of a band is observed around 1564 cm-1, indicating a change in the environment of the

guanine nucleotide owing to the loss of sRNAh structure, which occurs in the same temperature

region as observed in the other IR bands (Figure 3).

Small RNA hairpin base-sugar vibrations followed by FTIR spectroscopy

The base sugar vibrations were also followed by FTIR spectroscopy. The wavenumber area ranges

from 1500 to 1250 cm-1. In this region, the IR bands are originating from base-sugar vibrations

which are sensitive to glycosidic bond rotation, backbone and sugar pucker conformations. The IR

bands are related to backbone vibrations in A-, B- and Z-forms, sugar pucker in N- or S-type

conformations and N7 site vibrations of purines. It is a spectral region where a wide variety of

subbands can appear. However, the major IR bands in this spectra region cannot be unambiguously

assigned to the sRNAh.

Figure SI 6. Temperature-dependent normalized and second derivative spectra of the small RNA

hairpin in the wavenumber region from 1530 to 1380 cm-1. Second derivative and normalized

spectra of 5 mg mL-1 small RNA hairpin in 50 mM Tris-HCl buffer + 0.1 mM EDTA from 10 to

95 oC. IR bands are originating from base-sugar vibrations that are sensitive to glycosidic bond

rotation, backbone and sugar pucker conformations. The IR bands are related to backbone vibra-

tions in A-, B- and Z-forms, sugar pucker in N- or S-type conformations and N7 site vibrations of

purines. The most important bands are indicated by the grey rectangle in the second derivative

spectra. The highlighted bands are: 1522 cm-1, 1499 cm-1, 1476 – 60 cm-1, 1421 cm-1, 1402 cm-1.

If the RNA molecule adopts A-forms, i.e. right-handed helical conformations, the ribose sugar

adopts a C3'-endo conformation (the C3' atom of the sugar pucker is above the sugar plane and on

the same side as the base). The Z-form has the guanine sugar pucker in C3’-endo conformation

(syn position of G), but its cytosine stays in C2'-endo (C2' atom of sugar pucker is above the plane)

conformation (anti position of the C), which makes the (zig-zag) Z-form adopt a left-handed helical

conformation. C2'-endo sugar puckers are characteristic of right-handed B-form helices (anti po-

sition of nucleotides).

Figure SI 6 shows the temperature-dependent FTIR spectra of the sRNAh in the wavenumber re-

gion from 1530 to 1380 cm-1. The IR band at 1498 cm-1, which is assigned to in-plane vibrations

of cytosine, shows a significant decrease with temperature, which is most likely related to the shift

from base paired cytosine to free cytosine during denaturation of the sRNAh. An IR band can be

observed at 1403 cm-1, which is assigned to RNA vibrations of an in-plane C – O – H deformation

mode at the 2’ position of the ribose ring [2]. A small IR band at 1421 cm-1 related to purine

(guanine) sugar in S-type conformation (sugars at C2’-endo sugar pucker conformation of guanine

in B-form) can also be observed. An overall intensity increase in this wavenumber region indicates

conformational changes of sugar puckering of the sRNAh upon melting.

UV-Vis spectroscopy of the small RNA hairpin

In order to confirm the FTIR melting profile, UV-Vis spectroscopy measurements were carried

out. Nucleic acids are known to have a strong absorbance in the region of 240 – 275 nm. At neutral

pH, the UV absorption maxima range from 275 nm for guanine and 270 nm for cytosine to 260

nm for uracil. Polymeric RNA shows a broad absorbance around 260 nm. When nucleotides are

stacked, they are shielded from the solvent, and the absorbance is smaller compared to single

stranded nucleic acid chains. The RNA denaturation is then followed by an increase in the absorb-

ance around 260 nm.

A UV-1800 UV-Vis spectrometer from Shimadzu was used to collect absorbance spectra of the

small RNA hairpin. An external water bath connected to the cuvette holder kept the temperature

constant during all measurements. The measurements were carried out using standard quartz cu-

vettes with 300 µL of RNA sample volume. The labeled and non-labeled sRNAh samples were

lyophilized overnight and then diluted in 50 mM Tris-HCl buffer + 0.1 mM EDTA, to a final

concentration of 40 µM in 300 µL volume. The temperature varied from 10 to 80 oC in the cu-

vette. The sample temperature was equilibrated for 20 minutes before the acquisition of a new UV

spectrum.

The UV-Vis spectroscopy results displayed in Figure SI 5 are in good agreement with the results

from the FTIR data. They show an essentially continuous melting profile up to about 50 oC. With

increasing temperature, a shoulder appears in the UV-Vis spectra at 275 nm, which is related to

the absorbance of free guanine.

Figure SI 7. Temperature-dependent UV-Vis spectra of the small RNA hairpin. UV-Vis spectra

of 40 µM small RNA hairpin in 50 mM Tris-HCl buffer + 0.1 mM EDTA from 10 to 80 oC. The

UV-Vis absorbance increases with increasing temperature (indicated by the arrow).

Figure SI 8. Temperature-dependence of the maximum of the the UV-Vis absorbance at 259 nm

of the small RNA hairpin (40 µM small RNA hairpin in 50 mM Tris-HCl buffer + 0.1 mM EDTA

from 10 to 80 oC). The error bars cover the scattering of three independent measurements.

Labeled-small RNA hairpin in-plane base vibrations sensitive to effects of base pairing fol-

lowed by FTIR spectroscopy

Figure SI 9. Temperature-dependent normalized and second derivative spectra of the labeled -

small RNA hairpin in the wavenumber region from 1740 to 1600 cm-1. Second derivative and

normalized spectra of 5 mg mL-1 small RNA hairpin in 50 mM Tris-HCl buffer + 0.1 mM EDTA

from 10 to 80 oC. In the normalized spectra the IR band from 1696 to 1684 cm-1 is related to base

paired nucleotides and decreases with temperature (indicated by the arrow), while the IR band

from 1677 to 1653 cm-1 is related to free nucleotides and increases with temperature (indicated by

the arrow). The most important bands are indicated by the grey rectangles in the second derivative

spectra. The highlighted bands are: 1684 cm-1, 1675 cm-1, 1581 cm-1, 1653 cm-1, 1617-10 cm-1.

Figure SI 9 shows the temperature-dependent FTIR spectra of the labeled sRNAh in 50 mM Tris-

HCl buffer pH 7.5 + 0.1 mM EDTA, from 10 to 80 oC, in the wavenumber region from 1720 to

1600 cm-1. Thermal unfolding was detected by the decrease in absorbance of the IR band at 1686

cm-1, which is characteristic of base paired G-C vibrations, and the concomitant increase in IR

intensity of unpaired nucleic acids at around 1660 cm-1. The conformational changes observed in

the labeled-sRNAh have also been determined in terms of the ratio of intensity (Iratio) of unpaired

to paired G-C nucleotides pairs (Ifree/Iduplex) (Figure SI 10). These data were obtained from the IR

bands at 1660 and 1686 cm-1, respectively. The labeled sRNAh does neither show a "two state

model" like melting profile, nor is the melting process completed even at 80 oC. Hence, the labeled

sRNAh molecule presents a markedly different melting profile when compared to the unlabeled

sRNAh.

Figure SI 10. Ifree/Iduplex analysis of the temperature-dependent FTIR data of the labeled-small

RNA hairpin. Temperature-dependence of the ratio of unpaired to duplexed (GC pairs) ratio

(Ifree/Iduplex) as obtained from the infrared spectra recorded at about 1660 cm-1 and 1686 cm-1, re-

spectively, for 5 mg mL-1 labeled-small RNA hairpin in 50 mM Tris-HCl buffer + 0.1 mM EDTA

from 10 to 80 oC. The error bars cover the scattering of three independent measurements.

Small-angle X-ray Scattering studies on the labeled small RNA hairpin

All measurements were performed at the European Synchrotron Radiation Facility (ESRF), beam-

line ID 02 using a home-built high-pressure cell with diamond windows [3]. Samples of the labeled

sRNAh were prepared at a concentration of 20 mg mL-1 in 50 mM Tris-HCl-buffer+ 0.1 mM

EDTA, pH 7.5. The data was processed and background corrected using the software SAXSutili-

ties [4] provided by the beamline. Pair-distance distribution functions, P(r), were calculated using

the software GNOM, modelling using the software GASBOR of the ATSAS software package [5].

Figure SI 11. Influence of temperature and pressure on the overall shape of the labeled sRNAh.

Pair-distance distribution functions, P(r), in dependence of temperature (a) and pressure (b) and

models derived from the same data illustrating these changes (c). Temperature most likely pro-

motes the interaction of a fluorophore with the bases of the sRNAh.

The temperature-dependent P(r) functions in Figure SI 11a show an increase in distances, r, of

about 15 Å and a concomitant decrease of distances of about 30 Å with increasing temperature,

while the maximal dimension, rmax, does not change markedly, indicating a rearrangement within

the molecule. The pressure-dependent curves show an opposite trend, as can be seen in Figure

SI 11b. To resolve these findings in more detail, we generated models of the labeled sRNAh for

selected conditions (at 20 °C and 0.1 MPa, 60 °C and 0.1 MPa, 20 °C and 400 MPa). These models

reveal a dumbbell-shape of the labeled sRNAh with one handle larger than the other for 20°C at

0.1 MPa, see Figure SI 11c. The size of the larger handle of this dumbbell is in good agreement

with the shape and size of the folded gcUUCGgc sequence. The smaller handle probably reflects

the fluorophores linked to the latter. Upon temperature increase, the smaller handle becomes

smaller while the larger handle becomes lager. This could be interpreted by one fluorophore swap-

ping to the sequence region and forming a stacking interaction with the bases, explaining the

change in the stability towards temperature upon labeling. With pressure, both handles become

lager, suggesting partial unfolding of the labeled sRNAh with pressure.

Small RNA hairpin in-plane base vibrations sensitive to effects of base stacking followed by

FTIR spectroscopy

Figure SI 12. Pressure-dependent normalized and second derivative spectra of the small RNA

hairpin in the wavenumber region from 1600 to 1550 cm-1 at 20 oC. Second derivative and normal-

ized spectra of 5 mg mL-1 small RNA hairpin in 50 mM Tris-HCl buffer + 0.1 mM EDTA from

0.1 to 400 MPa at 20 oC. In the normalized spectra, the IR band at 1590 to 1575 cm-1 is related to

ring vibrations of C4 = C5 and C5 – C6 of free and base paired guanine and does not show signif-

icant changes. The IR band at 1568 to 1564 cm-1, related to ring vibrations of C6 = O6, C5 – C6

and C4 = C5 of free and base paired guanine, shows a small increase in intensity (indicated by the

arrow). The most important bands are indicated by the grey rectangle in the second derivative

spectra. The highlighted bands are: 1579 - 77 cm-1, 1566 - 63 cm-1.

Figure SI 12 shows the pressure-dependent FTIR spectra of the sRNAh from 0.1 to 400 MPa at 20 oC, in the wavenumber region from 1600 to 1550 cm-1. Both of these bands arise from ring vibra-

tions of free and base paired guanine. Changes in the position of this band are related to environ-

ment changes of guanine, and they are also used to follow the behavior of guanine-rich areas. The

intensity of both IRs band reduces when guanine forms base pairs. A very small increase of the IR

band around 1566 cm-1 observed, only. These results show that the effect of high hydrostatic pres-

sure even up to 400 MPa on the native structure of the sRNAh is rather small, especially when

related to guanine ring vibrations.

Small RNA hairpin base-sugar vibrations followed by FTIR spectroscopy

Figure SI 13. Pressure-dependent normalized and second derivative spectra of the small RNA

hairpin in the wavenumber region from 1540 to 1380 cm-1 at 20 oC. Second derivative and normal-

ized spectra of 5 mg mL-1 small RNA hairpin in 50 mM Tris-HCl buffer + 0.1 mM EDTA from

0.1 to 400 MPa at 20 oC. IR bands originate from base-sugar vibrations that are sensitive to glyco-

sidic bond rotation, backbone and sugar pucker conformations. The IR bands are related to back-

bone vibrations in A-, B- and Z-forms, sugar pucker in N- or S-type conformations and N7 site

vibrations of purines. The most important bands are indicated by the grey rectangle in the second

derivative spectra. The highlighted bands are: 1523 cm-1, 1500 cm-1, 1467 – 61 cm-1, 1403 cm-1.

Figure SI 13 shows the pressure-dependent FTIR spectra of the sRNAh from 0.1 to 400 MPa at 20 oC in the wavenumber region from 1540 to 1380 cm-1. The IR band at 1522 cm-1, which is charac-

teristic of in-plane vibrations of cytosine shows small variations in intensity, only. The IR band at

1500 cm-1, which is also characteristic of in-plane vibrations of cytosine, reveals a more significant

intensity decrease than the former band. This intensity decrease could be related to small structural

rearrangements caused by pressure. The small intensity changes in the low wavenumber region

(< 1425 cm-1) indicate also changes in sugar puckering upon compression. Hence, these observa-

tions are in agreement with the others IR bands observed in the IR spectra of the sRNAh, revealing

that at low temperatures, pressure has a small destabilizing effect on the sRNAh backbone and

sugar pucker conformation only when compared to the thermal denaturation profile.

Small RNA hairpin in-plane base vibrations sensitive to effects of base pairing followed by

FTIR spectroscopy

Figure SI 14. Pressure-dependent normalized and second derivative spectra of the small RNA

hairpin in the wavenumber region from 1740 to 1600 cm-1 at 70 oC. Second derivative and nor-

malized spectra of 5 mg mL-1 small RNA hairpin in 50 mM Tris-HCl buffer + 0.1 mM EDTA from

0.1 to 400 MPa at 70 oC. In the normalized spectra, the IR band from 1696 to 1684 cm-1, related

to base paired nucleotides, is not observed after denaturation. The IR band from 1677 to 1653 cm-

1, related to free nucleotides, does not show significant changes under high hydrostatic pressure.

The most important bands are indicated by the grey rectangle in the second derivative spectra. The

highlighted bands are: 1660 cm-1, 1658 cm-1, 1616 cm-1.

Small RNA hairpin in-plane base vibrations sensitive to effects of base stacking followed by

FTIR spectroscopy

Figure SI 15. Pressure-dependent normalized and second derivative spectra of the small RNA

hairpin in the wavenumber region from 1600 to 1550 cm-1 at 70 oC. Second derivative and normal-

ized spectra of 5 mg mL-1 small RNA hairpin in 50 mM Tris-HCl buffer + 0.1 mM EDTA from

0.1 to 400 MPa at 70 oC. In the normalized spectra, the IR band at 1590 to 1575 cm-1, related to

ring vibrations of C4 = C5 and C5 – C6 of free and base paired guanine, shows a significant

wavenumber shift (indicated by the arrow). The IR band at 1568 to 1564 cm-1, related to ring

vibrations of C6 = O6, C5 – C6 and C4 = C5 of free and base paired guanine, shows a small

intensity decrease with increasing pressure (indicated by the arrow). The most important bands are

indicated by the grey rectangle in the second derivative spectra. The highlighted bands are: 1586

- 81 cm-1, 1565 - 60 cm-1.

Figure SI 15 shows the pressure-dependent FTIR spectra of the sRNAh from 0.1 to 400 MPa at 70 oC in the wavenumber region from 1600 to 1550 cm-1. Both of these bands arise from ring vibra-

tions of free and base paired guanine. The intensity of both IR bands reduce when guanine forms

base pairs. It can be observed that the IR band around 1576 cm-1 shifts to higher wavenumber, to

~1582 cm-1, but the intensity does not change upon pressurization. Obviously, high hydrostatic

pressure does not have a significant effect on the ring vibrations of guanine of the sRNAh structure

at temperatures beyond the melting transition.

Small RNA hairpin base-sugar vibrations followed by FTIR spectroscopy

Figure SI 16. Pressure-dependent normalized and second derivative spectra of the small RNA

hairpin in the wavenumber region from 1560 to 1360 cm-1 at 70 oC. Second derivative and normal-

ized spectra of 5 mg mL-1 small RNA hairpin in 50 mM Tris-HCl buffer + 0.1 mM EDTA from

0.1 to 400 MPa at 70 oC. IR bands originate from base-sugar vibrations that are sensitive to glyco-

sidic bond rotation, backbone and sugar pucker conformations. The IR bands are related to back-

bone vibrations in A-, B- and Z-forms, sugar pucker in N- or S-type conformations and N7 site

vibrations of purines. The most important bands are indicated by the grey rectangle in the second

derivative spectra. The highlighted bands are: 1525 - 21 cm-1, 1500 cm-1, 1403 – 93 cm-1.

Figure SI 16 shows the pressure-dependent FTIR spectra of the sRNAh from 0.1 to 400 MPa at 70 oC, in the wavenumber region from 1540 to 1380 cm-1. The IR band at 1522 cm-1, which is char-

acteristic of in-plane vibrations of cytosine, shows a small shift to slightly higher wavenumber,

only. The IR band at 1500 cm-1, which is also characteristic of in-plane vibrations of cytosine,

displays a minor shift and intensity decrease. This intensity decrease may be related to a small

structural rearrangement caused by pressure, since the sRNAh is probably not fully unfolded.

High pressure FRET data on the labeled sRNAh

Figure SI 17. Pressure dependent FRET data of the sRNAh at selected temperatures from 10 to

70 oC. 24 µM sRNAh labeled at the 5' and 3' ends with Cy3 and Cy5 fluorescent dyes was prepared

in 0.1 mM EDTA and 10 mM phosphate buffer (pH 7.0).

Figure SI 17 combines the FRET data at various temperatures (from 10 to 70 0C) taken in the

pressure range from 0.1 to 275 MPa. These data show that even at temperatures below Tm (from

10 to 40 0C), pressure induces conformational changes. The continuous decrease of the FRET in-

tensity with increasing pressure points to an increasing destabilization of the native fold, or, in

other words, an increasing population of non-native conformations upon compression. The degree

of destabilization increases with increasing temperature, which is in agreement with the thermal

denaturation profile. Conversely, around and slightly above Tm (i.e., at 50 to 70 0C), pressure in-

crease leads to an increase of more compact, folded conformations of the sRNAh, suggesting a re-

entrant type of p,T-stability diagram of the sRNAh. At higher pressures, above 150 MPa, desta-

bilization of the compact structure sets in again. Interestingly, the degree of non-native, more open

conformational states of the sRNAh at the maximum pressure applied, 275 MPa, changes signifi-

cantly with temperature.

0 50 100 150 200 250 300

0.5

0.6

0.7

0.8

0.9

1.0

Inte

nsity /

a.u

Pressure / MPa

10 °C

20 °C

30 °C

40 °C

50 °C

55 °C

60 °C

70 °C

High pressure FTIR data on the labeled sRNAh

Figure SI 18. Pressure-dependent changes of the labeled-small RNA hairpin free base vibrations

of guanine and uracil followed by FTIR spectroscopy. Pressure-dependence change of the intensity

of unpaired GC pairs obtained from the infrared spectra recorded at about 1658 cm-1 for 5 mg mL-

1 labeled-small RNA hairpin in 50 mM Tris-HCl buffer + 0.1 mM EDTA from 0.1 to 400 MPa, at

20 oC and 70 oC.

Figure SI 18 reveals that the labeled-sRNAh structure is slightly stabilized by pressure, as can be

inferred from the observed small intensity decrease of single strand vibrations of guanines (1660

cm-1). The pressure-induced decrease of single strand intensity is more pronounced at 70 oC and

even a re-entrant phase behavior is observed. It has been shown by the temperature-dependent

FTIR and UV-Vis spectroscopy data that the labeled-sRNAh displays a different melting profile

compared to the unlabeled sRNAh. These results indicate that labeling of the sRNAh by the fluo-

rescent dyes Cy3 and Cy5 not only leads to a stabilization of the sRNA against temperature-in-

duced unfolding, but modulated also its pressure sensitivity markedly.

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