Supporting Information

Impact of spin label rigidity on extent and accuracy of distance information for PRE data

K. A. Schnorr1, D. B. Gophane2, C. Helmling1, E. Cetiner1, K. Pasemann1, B. Fürtig1,

A. Wacker1, N. S. Qureshi1, M. Gränz3, D. Barthelmes1, H. R. A. Jonker1, E. Stirnal1,

S. Th. Sigurdsson2, H. Schwalbe1*

1Institute of Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (BMRZ), Max-von-Laue Strasse 7, Johann Wolfgang Goethe-Universität, Frank-furt am Main, 60438, Germany 2University of Iceland, Department of Chemistry, Science Institute, Dunhaga 3, 107 Reyk-javik, Iceland3Institute of Physical and Theoretical Chemistry, Center for Biomolecular Magnetic Reso-nance (BMRZ), Max-von-Laue Strasse 7, Johann Wolfgang Goethe-Universität, Frankfurt am Main, 60438, Germany

*Address correspondence to: schwalbe@nmr.uni-frankfurt.de

Supplementary Table-S1 Excerpt of the computation for palindromicity of 10mer fragments for application in the 5’-truncated 68mer. The table illustrates the corresponding duplex interactions (secondary structure) with position of the canonical base pairs and the resulting Gibbs energies in kcal/mol. The secondary structure is illustrated in the dot-bracket representation. Brackets indicate a canonical Watson-Crick (AU; GC) and GU base pairing. A dot indicates a mismatch and the “&” character is the separator between the two oligomers.

The dot-bracket representation gives information about duplex formation. The brackets indi-cate only Watson-Crick (AU; GC) and GC base pairing. When no WC base pair interaction is possible, no brackets are shown. In case of oligomer number one, 10 WC base pairs can be formed by two 5’-AGUGGCCACU-3’, illustrated by 10 brackets. In case of oligomer number 954, only one WC base pair interaction is possible by two fragments. Hence, only one bracket is shown.

Supplementary Table S2 Overview of applied oligomers for non-covalent hybridization including the Gibbs energies for self-association (palindromicity) and the energies of the hybridization to the target sequence. The calculations with RNAduplex are based on the formation of canonical Watson-Crick (AU; GC) and GU wobble base pairing. Other effects, as potential Gquadruplex formation are not accounted for in the calculation.

Supplementary Figure S1 Schematic representation of the designed plasmids. Different plasmid building blocks including the cloning sites, the T7 promoter sequence, the corresponding ribozyme. (A) Schematic illustration for target construct 3’cut 64mer with 3’-HDV ribozyme (100 nt) for distinct truncation at the 3’-end. (B) Schematic illustration for target construct 5’cut 64mer with 5’-hammerhead ribozyme (99 nt) and 3’-HDV ribozyme (103 nt) for distinct truncation at both termini designed by Alexander Englert. (C) Scheme for target construct 3’cut 68mer with 3’-HDV ribozyme (100 nt) for distinct truncation at the 3’-end. (D) Schematic representation for target construct 5’cut 68mer with 5’-hammerhead ribozyme (99 nt) for distinct truncation at the 5’-end.

Besides the performed non-covalent spin labeling approach, chemically synthesized RNA can be stable isotope labeled and hence also be used for PRE determination when equipped with a spin label, as described by Wunderlich et al. (2013).

Supplementary Figure S2 CW-EPR spectra, visualizing the integrals of different spin systems. The spectra were normalized on the central dispersive signal and measured at room temperature. (A) CW-EPR spectra of the TEMPO group alone and covalently bound to the 10mer fragment. (B) CW-EPR spectra of the TEMPO10mer fragment and in complex with 68mer forming the TEMPO78mer construct. (C) CW-EPR spectra of the Çm group alone and covalently bound to the 10mer fragment. (D) CW-EPR spectra of the Çm10mer fragment and in complex with 68mer RNA forming the Çm78mer system.

Supplementary Table S3 Values of rotational correlation time τr for the different spin systems determined by EasySpin least squares fitting (Stoll and Schweiger 2006).

CW-EPR analysis to investigate changes in dynamics of τc of the electron upon titration of ligand and heating.

Supplementary Figure S3 CW-EPR spectra of sample 68mer + Cm10mer (A) and 68mer + TEMPO10mer (B). Integrals marked in red show the complexes alone; blue: after addition of 2 equiv of 2’dG and 8 equiv of Mg2+; orange: after addition of 2 equiv of 2’dG and 8 equiv of Mg2+ and a heating step for 4 min @ 65 °C. Asterisks indicate artifacts from sample tubes.

Supplementary Figure S4 (A) 1H-15N-TROSY spectrum recorded in the diamagnetic state on the I-A 68 + TEMPO10mer at 800 MHz. (B) 13C-filtered NOESY spectrum recorded in the diamagnetic state on the I-A 68 + TEMPO10mer with a mixing time of 150 ms at 600 MHz. D: 1H-13C-HSQC spectrum recorded in the diamagnetic state on the I-A 68 + TEMPO10mer at 800 MHz. Spectra A,B,D were recorded on the diamagnetic state on the I-A 68 + TEMPO10mer on a 350 µM 68mer sample with 1.2 equiv of oligomer and 2 equiv of 2’dG in NMR buffer at 283 K. C: Secondary structure of the 3’cut-I-A 68 + TEMPO10mer RNA illustrating the applied isotope labeling scheme: grey = unlabeled, blue = 15N labeled, green = 15N, 13C labeled nucleotides.

Supplementary Figure S5 Overlay of 1H-13C-HSQC of IA-70mer, stabilized wild-type (blue) and 68mer + TEMPO10mer, (diamagnetic state, black) at 800 MHz. The assignment in black corresponds to the P1-elongated 68mer + TEMPO10mer construct. Orange labels indicate nucleotides that are either not present in the wild-type or fundamentally shifted due to sequential alterations in the two aptamers.

Supplementary Figure S6 (A) 1H-15N-TROSY spectrum recorded in the diamagnetic state on the I-A 68 + Çm10mer at 800 MHz. (B) 13C-filtered NOESY spectrum recorded in the diamagnetic state on the I-A 68 + Çm10mer with a mixing time of 150 ms at 600 MHz. (D) 1H-13C-HSQC spectrum recorded in the diamagnetic state on the I-A 68 + TEMPO10mer at 800 MHz. Spectra (A), (B), (D) were recorded on the diamagnetic state on the I-A 68 + Çm10mer on a 350 µM 68mer sample with 1.2 equiv of oligomer and 2 equiv of 2’dG in NMR buffer at 283 K. (C) Secondary structure of the 5’cut-I-A 68 + Çm10mer RNA illustrating the applied isotope labeling scheme: grey = unlabeled, blue = 15N labeled, green = 15N, 13C labeled nucleotides.

Supplementary Figure S7 Overlay of 1H-13C-HSQC of IA-70mer, stabilized wild-type (blue) and 68mer + Çm10mer, (diamagnetic state, black) at 800 MHz. The assignment in black corresponds to the P1-elongated 68mer + Çm10mer construct. Orange labels indicate nucleotides that are either not present in the wild-type or fundamentally shifted due to sequential alterations in the two aptamers.

Supplementary Table S4 Potential and effectively used PRE restraints for both applied spin labels and the different detection approach. Theoretical restraints result from the number of isotope labeled Gua-H1, Uri-H3, and Ade-H2 protons. Marked with n.d. gives the numbers of resonances which were omitted due to spectral overlap, or ambiguous assignment. S/N corresponds to signals which showed too low S/N for reliable analysis.

System for PREDetermination

theoreticalrestraints

n.d. effectivelyused restraints

S/N

UTM (1HN) 32 2 30 0UTM (1HC) 19 12 6 1Çm (1HN) 32 7 25 0Çm (1HC) 20 7 9 4

C40/U41C43

U77

U55

U27

U45

U20

U68

Supplementary Figure S8 Secondary structure of the I-A aptamer. The colored lines represent possible ligation sites for different spin labeling positions. It is desired to keep the 2’-amino modified RNA as short as possible to increase ligation yield and minimize the number of unlabeled nucleotides. The arrows point into the directions of each unlabeled 2’-amino modified RNA.

Supplementary Table S5 Summary of calculated proton distances for structure prediction. Empty boxes represent zero values and were omitted for the sake of clarity.

Spin label position

Incorpo-ration of ligation

suggestion

Quantity of 15N-protons ≥ 14 Å

≤ 23 Å

Quantity of 13C-protons ≥ 14 Å

≤ 23 Å

Quantity of 15N-protons < 14 Å

Quantity of 13C-protons < 14 Å

P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3

U27 No 8 6 3 3U27 Yes 7 4 1U41 No 7 3 2 4 5U41 Yes 3 2U45 No 6 3 6 4 3U45 Yes 4 2 4 3 3U55 No 1 1 3 2 1 4 4 2 1U55 Yes 1 1 1 2U77 No 6 4 1 3 3 8 2U77 Yes 4 4 3 2 5 2U20 Yes 3 4 2 2 1 4 2U68 Yes 3 2 1 3 1 5C40 No 8 6 2 3 4C40 Yes 3 3 2 2C43 No 5 5 5 4C43 Yes 4 5 5 4

Supplementary Table S6 Experimental and simulated PRE rates for system I-A68 + TEMPO-10mer. d: PRE rate < 5 Hz; corresponding distance restraint > 23 Å. e: PRE rate estimated > 99 Hz, due to fully bleached resonance in para; corresponding distance restraint < 14 Å.

Supplementary Table S7 Experimental and simulated PRE rates for system I-A68 + Çm-10mer. d: PRE rate < 5 Hz; corresponding distance restraint > 23 Å. e: PRE rate estimated > 99 Hz, due to fully bleached resonance in para; corresponding distance restraint < 14 Å.

Synthesis of the rigid spin label Çm. Rigid spin label Çm and its phosphoramidite was pre-

pared by previously reported procedure (Höbartner et al. 2012). Çm was incorporated into

RNA oligoribonucleotides by solid phase synthesis using previously reported protocols

(Cekan and Sigurdsson 2008; Höbartner et al. 2012). Upon completion of the synthesis (1 µmol

scale), the oligoribonucleotides were cleaved from the solid support and the nucleobases and the

phosphodiesters deprotected in a 1:1 mixture of conc. aqueous NH3 and 8 M MeNH2 in EtOH (2

mL) at 65 °C for 40 min. The supernatant was collected, the beads washed three times with a

mixture of EtOH : water (1:1, 300µL), and the combined washes dried. The 2′-O-TBDMS

groups were removed by treatment with a mixture of Et3N•3HF:DMF (3:1, 800 µL) at 55 °C for

1.5 h, followed by addition of H2O (200 µL). This mixture was transferred to a 50 mL Falcon

tube and n-butanol (40 mL) added and stored at Excess Et3N•3HF was quenched by addition of

water and the oligoribonucleotides were precipitated with n-butanol at -20 °C for 12 h, centri-

fuged and the solvent decanted from the RNA pellet. After drying the RNA was purified by

HPLC, followed by 4 runs of lyophilisation to remove residual Triethylammonium acetate.

References for Supplementary Material

Cekan P, Sigurdsson ST (2008) Single base interrogation by a fluorescent nucleotide: each of the four DNA bases identified by fluorescence spectroscopy. Chem Commun (Camb) 3393–3395. doi: 10.1039/b801833b

Höbartner C, Sicoli G, Wachowius F, et al (2012) Synthesis and characterization of RNA con-taining a rigid and nonperturbing cytidine-derived spin label. J Org Chem 77:7749–54. doi: 10.1021/jo301227w

Stoll S, Schweiger A (2006) EasySpin, a comprehensive software package for spectral simula-tion and analysis in EPR. J Magn Reson 178:42–55. doi: 10.1016/j.jmr.2005.08.013

Wunderlich CH, Huber RG, Spitzer R, et al (2013) A Novel Paramagnetic Relaxation Enhance-ment Tag for Nucleic Acids: A Tool to Study Structure and Dynamics of RNA. ACS Chem Biol. doi: 10.1021/cb400589q