Unity inova 700 mhz spectrometer

¹H-NMR spectra were acquired at 5, 25, 37 and 49 °C either on a Varian Unity Inova 700 MHz spectrometer equipped with an HCN triple resonance cryoprobe or on a Varian Unity INOVA 500 MHz spectrometer equipped with a broadband inverse probe with z-field gradient and processed using the Varian VNMR and iNMR (http://www.inmr.net) software packages. All micro RNA samples were prepared at ~0.3 mM concentration by dissolving 75 nmol of each miRNA in 250 µL of 10 mM PBS buffer at pH 6.8. The spectra were acquired as 16,384 data points with a recycle delay of 1.0 s; data sets were zero-filled to 32,768 points prior to Fourier transformation and apodized with a shifted sine bell squared window function. Water suppression was achieved by including a double pulsed-field gradient spin-echo (DPFGSE) module^{45 (link),46 (link)} in the pulse sequence prior to acquisition.

Chemicals and solvents were purchased from Sigma-Aldrich (Milan, Italy). Reagents and phosphoramidites for DNA syntheses were purchased from Glen Research (Sterling, VA, USA). The DEL linker 2, the TEL linker 3 and all the ODNs were assembled on a PerSeptive Biosystems (Framingham, MA, USA) Expedite DNA synthesizer using standard phosphoramidite chemistry. HPLC analyses and purifications were performed with a Jasco (Jasco Europe Srl, Cremella, LC, Italy) PU2089 pump system equipped with an UV detector model 2075 Plus. CPG-resin 1 and the linkers 4 and 5 were purchased from Glen Research. UV spectra were registered using a Jasco V-530 spectrophotometer. CD spectra and thermal denaturation experiments were obtained with a Jasco 1500 CD spectrophotometer equipped with a Jasco PTC-348-WI temperature controller unit. NMR spectra were recorded either on a Varian (Palo Alto, CA, USA) Unity Inova 500 MHz spectrometer equipped with a broadband inverse probe with z-field gradient, or on a Varian Unity INOVA 700 MHz spectrometer equipped with a triple resonance cryoprobe. All NMR spectra were processed using the Varian VNMR and iNMR (http://www.inmr.net) software packages. PAGE bands were visualized on a Bio-Rad Laboratories (Segrate, MI, Italy) Gel Doc^TM XR+ image system.

The solution structures for NMR analysis of RiLK1 and 1018-K6 peptides were determined under different conditions: pure water (H₂O/D₂O 90/10 v/v) pH 4 and in micellar SDS (H₂O/SDS-d25 150 mM, pH 4.4). Deuterated water (98% isotopic purity) and SDS-d25 (98% isotopic purity) were purchased by Sigma Aldrich, Milano, Italy. NMR spectra were recorded on a Varian Unity Inova 700MHz Spectrometer located at the Department of Pharmacy—University of Naples “Federico II”. The 2D 1H-NMR, TOCSY (mixing time 70 ms) and NOESY (mixing time 300 ms) were recorded at 298 K. The water resonance was suppressed by the use of gradients. Chemical shifts were referred to internal sodium 3-(trimethylsilyl) propionate 2,2,3,3-d4 (TSP, Sigma Aldrich, Milano, Italy). NMR spectra were analyzed by using CARA program (http://cara.nmr.ch/doku.php/home, accessed on 10 March 2021). Proton resonances were sequentially assigned by following the Wuthrich standard method [61 ].
NMR data were deposited in the Biological Magnetic Resonance Bank (BMRB entry 50902).