Supplementary Material

Development and Application of Aromatic [13C, 1H] SOFAST-HMQC NMR Experiment for Nucleic Acids

Bharathwaj Sathyamoorthy1, Janghyun Lee1,2, Isaac Kimsey1, Laura Ganser1, Hashim Al-Hashimi1*

1Department of Biochemistry, Duke University, Durham, NC 27710, USA

2 Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA

E-mail:

I. Materials and methods

Sample preparation

TAR: Uniformly 13C/15N labeled TAR RNA was prepared by in vitro transcription using double stranded DNA encoding the RNA sequence of interest and containing the T7 promoter at 5´-end (Integrated DNA Technologies). T7 RNA polymerase (Takara Mirus Bio, Inc.) was used to transcribe the DNA sequence in the presence of 13C/15N labeled nucleotide triphosphates (ISOTEC, Inc. and Cambridge Isotope Laboratories, Inc). The RNA was purified using 20% (w/v) denaturing polyacrylamide gel electrophoresis (PAGE) in 8 M urea and 1X TBE. The RNA was electro-eluted in 20mM Tris (pH 8) buffer and then precipitated in ethanol. The purified RNA pellet was dissolved and exchanged and concentrated in the NMR buffer (15mM NaH2PO4/Na2HPO4, 25mM NaCl, 0.1mM EDTA, 10% (v/v) D2O, and pH ~ 6.4) using a 3 kDa centrifugal concentrator (Millipore Corp.) to a final concentration of 1 mM. 1.5 μL of 20mM capreomycin (in 100% H2O, purchased from MP Biomedicals and used as is) was added to 500 μL of 10 μM uniformly13C/15N labeled TAR in the NMR buffer to achieve the molar ratio of 1:6 for TAR to capreomycin.

tRNAPhe: Primary sequence - 5´-GCGGATTTAGCTCAGTTGGGAGAGCGCCAGACTGAAGATCTGGAGGTCCTGTGTTCGATCCACAGAATTCGCACCA -3´

Protocol for tRNAPhe sample was preparation is as described for TAR. The purified RNA pellet was dissolved and exchanged and concentrated in the NMR buffer (10 mM NaH2PO4/Na2HPO4, 100 mM NaCl, 20 mM MgCl2, 10% (v/v) D2O, and pH ~ 6.5) using a 3 kDa centrifugal concentrator (Millipore Corp.) to a final concentration of ~0.5 mM.

DD-dsDNA: The oligo was purchased from Integrated DNA Technologies and was used without further purification. The oligo was annealed at 95 °C for 5 minutes followed by room temperature for 30 minutes. The sample was then buffer exchanged and concentrated using a 3 kDa centrifugal concentrator (Millipore Corp.) to a final duplex concentration of ~ 3.5 mM, the buffer containing 15mM NaH2PO4/Na2HPO4, 25 mM NaCl, 0.1mM EDTA, 10% (v/v) D2O, and pH ~ 6.8.

DD-GT-dsDNA: The oligo was synthesized using the protocol established by Zimmer and Crothers (Proc Nat Acad Sci USA, 1995, 92, 3091-3095). Synthesis was carried out at 14 mL scale with 0.1 U / μL of exonuclease deficient Klenow polymerase and 15 mM MgCl2. Polymerase was heat-inactivated after 24-hours at 37 °C. Target oligo was cleaved from template strand via alkaline hydrolysis of the rU (55 °C for 3 hours in 0.3M NaOH). Hydrolyzed reaction mixture was concentrated to 1 mL and separated using a 20% polyacrylamide gel. Target band was excised and oligo was extracted using standard “crush and soak” procedure followed by ethanol precipitation. The target oligo was then buffer exchanged and concentrated using a 3 kDa centrifugal concentrator(Millipore Corp.) with the NMR buffer (125 mM NaCl, 15 mM KH2PO4/K2HPO4, 0.1 mM EDTA10% (v/v) D2O, and pH ~ 6.8) to a final duplex concentration of 3 mM.

Optimization of Ernst angle for aromatic protons at short inter-scan delays

Fig S1.Signal intensity of aromatic protons for tRNAPhe RNA collected from the first FID of a SOFAST-HMQC experiment as a function of the excitation flip angle for a recycle delay of 250 ms. The maximum sensitivity was observed to be ~120°, similar to TAR and DD-GT-dsDNA.

NMR data acquisition

Table S1. SOFAST-HMQC data acquisition parameters

Sample / Concentration / Experiment / Recycle delay (ms) / Number of scans / spectral widths 1H;13C/15N (ppm) / number of real points 1H;13C/15N / carrier position 13C/15N (ppm) / selective pulse carrier/ bandwidth (ppm) / Total measurement time / Fig in text
TAR / 1 mM / aromatic / 200, 250,..., 950, 1000, 1250, 1500, 1750, 2000 / 8 / 10.0/10.0 / 512/96 / 140.6 / 8.0/3.0 / 8.2 hours / 1C
TAR / 1 mM / aromatic / 50 / 2 / 10.0/10.0 / 256/48 / 140.6 / 8.0/3.0 / 19 s / 2A
TAR / 10 μM / aromatic / 500 / 144 / 10.0/10.0 / 512/134 / 140.6 / 8.0/3.0 / 6 hours / 2B
TAR + Capreomycin / aromatic
TAR / 1 mM / long-range 15N-1H / 500 / 4 / 22.0/100.0 / 1024/233 / 200 / 8.0/3.0 / 20 mins / 3A
tRNAPhe / 0.5 mM / aromatic / 150, 200, 250, 300, 400,..., 900, 1000, 1250, 1500, 1750, 2000 / 4 / 10.0/24.5 / 512/120 / 146.1 / 8.0/3.0 / 4.0 hours / 1D
tRNAPhe / 0.5 mM / aromatic / 75 / 2 / 10.0/24.5 / 256/60 / 146.1 / 8.0/3.0 / 32 s / 2C
DD-dsDNA* / 3.5 mM / aromatic / 500 / 2 / 10.0/10.0 / 512/64 / 140.6 / 8.0/3.0 / 210 s / 2D
DD-dsDNA* / 3.5 mM / long-range 13C-1H / 250 / 2048 / 22.0/10.0 / 512/32 / 115.1 / 13.1/4.0 / 13 hours / 3B
DD-GT-dsDNA / 3.0 mM / aromatic / 200, 250,..., 950, 1000, 1250, 1500, 1750, 2000 / 4 / 10.0/2.8 / 512/36 / 139.1 / 8.0/3.0 / 1.8 hours / 1E
DD-GT-dsDNA / 3.0 mM / long-range 13C-1H / 250 / 2 / 22.0/10.0 / 512/32 / 115.1 / 12.0/6.0 / 60 s / 3B

II. Sparse sampling prospective:

Fig S2. The 2D [13C, 1H] aromatic SOFAST-HMQC spectrum obtained by processing the uniformly sampled 2D data with (A, B) Fourier Transformation (FT) and (C-E) Compressed Sensing (CS). (A) Same as Fig 2A from main text. (B) High resolution and higher sensitivity conventional HMQC for comparison (parameters Table S1, row 1). (C), (D) and (E) CS processed spectrum with 100%, 75% and 50%, respectively, of the indirect 13C dimension evolution points. The sparsely sampled data is chosen based on a Poisson Gap distribution. The quality of the 2D spectra illustrate that sparsely sampling the indirect evolution points to 50% can reproduce the spectra obtained with uniform sampling and FT. Reducing the sampling further causes the weak C2-H2 peaks to disappear, which can be improved by optimizing between the percentage of sparse sampling and inter-scan delay.