Observing an Antisense Drug Complex in Intact Human Cells by in‐Cell NMR Spectroscopy

Abstract

Gaining insight into the uptake, trafficking and target engagement of drugs in cells can enhance understanding of a drug's function and efficiency. However, there are currently no reliable methods for studying untagged biomolecules in macromolecular complexes in intact human cells. Here we have studied an antisense oligonucleotide (ASO) drug in HEK 293T and HeLa cells by NMR spectroscopy. Using a combination of transfection, cryoprotection and dynamic nuclear polarization (DNP), we were able to detect the drug directly in intact frozen cells. Activity of the drug was confirmed by quantitative reverse transcription polymerase chain reaction (qRT‐PCR). By applying DNP NMR to frozen cells, we overcame limitations both of solution‐state in‐cell NMR spectroscopy (e.g., size, stability and sensitivity) and of visualization techniques, in which (e.g., fluorescent) tagging of the ASO decreases its activity. The capability to detect an untagged, active drug, interacting in its natural environment, represents a first step towards studying molecular mechanisms in intact cells.

Aromatic 19F–13C TROSY—[19F, 13C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA

“…20 kD) ( Figure 1d). We further incorporated a [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] Our initial efforts focused on the production of the [5-19 F, 5-13 C]-uridine phosphoramidite 7.B ased on our recently published synthetic access to a5-13 C-uridine building block [9] we included af luorination step using the commercially available fluorination reagent Selectfluor TM .T he synthetic route starting from [5-13 C]-uracil is shown (Scheme 1). The compound 1 was produced from [2-13 C]-bromoacetic acid, potassium cyanide and urea as previously described.…”

“…As the first target, the 30 nt HIV TAR-2 RNAwas chosen as for this system 19 FNMR assignment data for an in vitro transcribed [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] F]-uridine labeled version is available. [2c] All six uridines were substituted by the [5-19 F, 5-13 C]-counterparts ( Figure 2a).…”

“…Schwalbe and coworkers used the latter method to probe the effect of all 2-F-Al abeling in the 73 nt guanine sensing riboswitch from bacillus subtilis. [3a] Only four resonances of sixteen could be assigned illustrating that in larger RNAs resonance assignment issues arise.Thus,anexpansion of the 19 Fstable isotope labeling scheme to yield a 19 F- 13 C-spin topology,such as a [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] F, 5-13 C]-labeling pattern in pyrimidines,would be desirable to evade resonance overlap issues in larger RNAs by adding asecond 13 Cdimension. Theapplication of 19 F-NMR to study high molecular weight systems is also hampered by the fast transverse relaxation by the chemical shift anisotropy (CSA) mechanism.…”

“…In ar ecent work this issue was addressed by introducing aromatic 19 F- 13 C-spin topologies in proteins and DNA, in which the cancellation of the dipole-dipole interaction and the chemical shift anisotropy relaxation mechanisms lead to avery favorable NMR spectroscopic behavior in aTROSY type experiment. [6] Here,wereport on the synthetic access to [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] F, 5-13 C]-uridine (U) and -cytidine (C) phosphoramidites for solid phase RNAsynthesis.W edemonstrate the favorable spectroscopic behavior of the [5-19 F, 5-13 C]pyrimidines in two model RNAs:the 61 nt human hepatitis B virus encapsidation signal epsilon (hHBV e)a nd the 59 nt pre-microRNA21. [7] Thefeasibility of the 13 C/ 19 Flabeling by solid phase RNAsynthesis is shown and used for aresonance assignment protocol based on [5-19 F, 5-13 C]/[5-19 F]-uridine labeling schemes.F or the 61 nt hHBV e RNAw eo bserved afavorable TROSY effect and compared the performance of [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] F, 13 C]-U labels with [6-1 H, 6-13 C]-U labels.T he novel stable isotope (SI) labeling scheme will facilitate NMR studies of large RNAs and high molecular weight RNA protein complexes as it was recently demonstrated for high molecular weight proteins systems.…”

Aromatic 19F–13C TROSY—[19F, 13C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA

“…Forschungsartikel phosphoramidites 7 and 11 were available RNAs with sizes between 30 and 60 nts were synthesized. First, the 30 nt HIV TAR-2 with six uridines and the 61 nt hHBV e RNAw ith eighteen uridines were synthesized and six and five [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] F, 5-13 C]-U labels were incorporated in each RNA, respectively. We obtained high quality 19 F- 13 CT ROSY spectra within as hort time on aP rodigy TCI probe with the proton coil tuned to the 19 Fr esonance frequency for 0.5 mm RNA samples.T he additional 13 Cd imension allowed to resolve the resonance overlap,w hich was earlier observed for U23, U25 and U31 in the 1D-19 Fs pectrum.…”