M mulv enzyme

Uniformly {¹³C, ¹⁵N}-labeled KRAS 32R WT DNA was purchased from Silantes GmbH. G9T and G25T samples were produced in our lab using M-MuLV enzyme from New England Biolabs and dNTPs from Eurisotop. Non-labelled samples were purchased from IDT (Integrated DNA Technologies; USA). They were synthesized on the 250 nmol scale and purified by reverse-phase HPLC. Samples labelled site-specifically at a level of 5% ¹⁵N, ¹³C were synthesized in our laboratory (INSERM U1212, Bordeaux) on an automated Expedite 8909 DNA synthesizer at 1 μmol scale on a 1000-Å primer support (Link Technologies SynBase CPG). All the standard phosphoramidites (dABz, dT, dGiBu, dCAc), reagents, and solvents used during the synthesis were purchased from Glen Research. The dGiBu-phosphoramidite (U-¹³C10, 98%; U-¹⁵N5, 98%; CP 95%) was purchased from Cambridge Isotope Laboratories. After the synthesis, the oligonucleotides were cleaved from the support and the nucleobases were deprotected with ammonium hydroxide at 55°C for 16 h and then lyophilized. Oligonucleotide solutions were prepared in 1× buffer (10 mM K₂HPO₄/KH₂PO₄; 50 mM KCl; pH 6,6). In order to form G-quadruplexes, samples are heated at 95°C for 5 min then quickly cooled in ice. This process was repeated four times and the final sample kept at 4°C. All NMR samples presented in the manuscript were re-synthesized and each spectra repeated more than once.

CAR-T cells and control T cells were lysed in TRIzol reagents (Life Technologies) and frozen at −80°C. Total RNA extraction was performed according to the manufacturer’s instructions. Reverse transcription of cDNA was performed using M-Mulv enzyme (NEB), and qPCE was performed using 2 × SYBR Green RT-PCR Mix. Expression analysis of mRNA was normalized to β-actin, and data were analyzed by applying the 2^(-dCt) calculation method.