Spin-Labeled Riboswitch Synthesized from a Protected TPA Phosphoramidite Building Block

Abstract

The nitroxide TPA (2,2,5,5-tetramethyl-pyrrolin-1-oxyl-3-acetylene) is an excellent spin label for EPR studies of RNA. Previous synthetic methods, however, are complicated and require special equipment. Herein, we describe a uridine derived phosphoramidite with a photocaged TPA unit attached. The light sensitive 2-nitrobenzyloxymethyl group can be removed in high yield by short irradiation at 365 nm. Based on this approach, a doubly spin-labeled 27mer neomycin sensing riboswitch was synthesized and studied by PELDOR. The overall thermal stability of the fold is not much reduced by TPA. In-line probing nevertheless detected changes in local mobility.

Keywords: PELDOR; RNA aptamer; nitroxide; photocaged spin label; secondary structure mapping.

Figure 1

Phosphoramidite building blocks containing protected TEMPO (1) and TPA (3) spin labels. The resulting final RNA structures 2 and 4 are shown below.

Scheme 1

Synthesis of phosphoramidite 3 containing a protected TPA spin label. a) Br₂, HOAc, 85 %; b) N,O‐dimethylhydroxylamine hydrochloride, Et₃N, H₂O, 85 %; c) m‐chloroperbenzoic acid, CH₂Cl₂, 84 %; d) DMSO, Ac₂O, AcOH, then aqueous NaOH, 72 %; e) 10, SO₂Cl₂, then addition of 8, Cu, Cu(OTf)₂, 4,4’‐dimethyl‐2,2‐bipyridyl, toluene, Δ, 52 %; f) DIBAL, Et₂O, toluene, 91 %; g) dimethyl (1‐diazo‐2‐oxopropyl)phosphonate, MeOH, K₂CO₃, 46 %; h) Ac₂O, DMAP, Et₃N, AcCN, 89 %; i) I₂, (NH₄)₂[Ce(NO₃)₆], AcCN, 88 %; j) NaOMe, MeOH, 97 %; k) 4,4’‐dimethoxytrityl chloride, pyridine, 84 %; l) tert‐butyldimethylsilyl chloride, DMF, imidazole, 32 %; m) 13, (Ph₃P)₄Pd, CuI, Et₃N, DMF, 78 %; n) N,N‐diisopropylaminocyanoethylphosphor amidic chloride, Et₃N, CH₂Cl₂, 86 %.

Figure 2

Structure of the palindromic RNAs 21–23, here shown in form of a duplex. In compound 22, the TPA group is protected by the 2‐NBOM group. Irradiation then leads to hemiacetal 22 a that eliminates CH₂O upon heating and forms nitroxide 23 by spontaneous air oxidation. Significant amounts of amine 24, a possible byproduct that cannot be reoxidized, were not observed.

Figure 3

PELDOR measurement of RNA 23. Left panel: PELDOR time trace after background correction (for original data, see Supporting Information) and fit with DEERNet (red) by DEER analysis. Right panel: Distance distribution with a maximum at 3.94 nm.

Figure 4

Variants of the neomycin‐responsive riboswitch prepared for this study (for details of structure see Supporting Information). RNA 25 was used for EPR spectroscopy. The dye‐labeled analogs 27 and 29 served as samples for in‐line probing to identify the conformational impact of the TPA labels. The secondary structures are represented as in a previous publication. However, in the unmodified RNA 27, according to in‐line probing, only nucleotides printed in red show increased mobility.

Figure 5

PELDOR measurement of aptamer RNA 25. Left panel: PELDOR time trace after background correction (for original data, see Supporting Information) and fit with DEERNet (red) by DEER analysis. Right panel: Distance distribution with a maximum at 3.4 nm (sample briefly annealed at 0 °C before freezing).

Figure 6

Secondary structure mapping of riboswitch RNAs 27 and 29 by in‐line probing. A: Incubation of RNA 27 at 37 °C (20 h, 20 mM Mg²⁺). Not much cleavage occurs in positions 9–13 indicating the presence of additional base pairs. B: Incubation of RNA 29 at 37 °C (20 h, 20 mM Mg²⁺). The central loop opens up. C: Incubation of RNA 29 at 4 °C (110 h, 20 mM Mg²⁺).