Ethylenediamine derivatives efficiently react with oxidized RNA 3' ends providing access to mono and dually labelled RNA probes for enzymatic assays and in vivo translation

Abstract

Development of RNA-based technologies relies on the ability to detect, manipulate, and modify RNA. Efficient, selective and scalable covalent modification of long RNA molecules remains a challenge. We report a chemical method for modification of RNA 3'-end based on previously unrecognized superior reactivity of N-substituted ethylenediamines in reductive amination of periodate-oxidized RNA. Using this method, we obtained fluorescently labelled or biotinylated RNAs varying in length (from 3 to 2000 nt) and carrying different 5' ends (including m7G cap) in high yields (70-100% by HPLC). The method is scalable (up to sub-milligrams of mRNA) and combined with label-facilitated HPLC purification yields highly homogeneous products. The combination of 3'-end labelling with 5'-end labelling by strain-promoted azide-alkyne cycloaddition (SPAAC) afforded a one-pot protocol for site-specific RNA bifunctionalization, providing access to two-colour fluorescent RNA probes. These probes exhibited fluorescence resonance energy transfer (FRET), which enabled real-time monitoring of several RNA hydrolase activities (RNase A, RNase T1, RNase R, Dcp1/2, and RNase H). Dually labelled mRNAs were efficiently translated in cultured cells and in zebrafish embryos, which combined with their detectability by fluorescent methods and scalability of the synthesis, opens new avenues for the investigation of mRNA metabolism and the fate of mRNA-based therapeutics.

Figure 1.

Reductive amination of GMP-dial with different N-nucleophiles (1–10). (A) Reaction scheme with general structure of reductive amination products GMP-(n). (B–D) HPLC chromatograms of crude reaction products after 40 min (1–6, 8) or 60 min (7, 9, 10) after addition of the nucleophile. During reaction between GMP-dial and butylamine (2) or cysteamine (7) indicated morpholine products (GMP-2 and GMP-7) were not detected. Product of reductive amination between GMP-dial and ethylenediamine (10, EDA) is designated as GMP-10 or GMP-EDA.

Figure 2.

One-pot 3’-end RNA labelling. (A) Reaction scheme. Conditions: i) pU₃ (140 µM), aqueous NaIO₄ (1.4 mM), 30 min, and 25°C in the dark. ii) Addition of R-EDA (1.0 mM) and NaBH₃CN (20 mM) in KH₂PO₄ buffer (0.1 M pH 6.0). HPLC-determined yields after two hours of reductive amination are presented for each N-nucleophile. (B) HPLC chromatograms of reaction with Cy3-EDA at different stages. (C) Second-order reaction rate constant k values determined for reductive amination reaction between pU3-dial (100 µM) and different N-nucleophiles (1 mM).

Figure 3.

Mono- and dual labelling of IVT RNA. (A) Scheme of the process: NTPs mix in the presence or absence of initiating dinucleotide (ARCA, N₃-AG or N₃-m⁷GpppG) are incubated with gene-coding DNA template in presence of T7 RNA polymerase. After isolation from IVT mixture, RNA products are subjected to 3′ labelling (top) or simultaneous 5′+3′ dual labelling (bottom). (B) Representative HPLC chromatograms of crude labelling products of Cy3 3′ labelling (top) or Cy5/Cy3 5′+3′ dual labelling (bottom) performed on 35, 276 and 993 nucleotide-long RNAs (ppp-RNA₃₅, N₃-RNA₃₅, N₃-m⁷GRNA₂₇₆ and N₃-m⁷GRNA_gluc). (C) Absorbance and fluorescence HPLC profiles of crude dual labelling product Cy5-m⁷GRNA_gluc-Cy3 with designated retention times of collected fractions (right) and agarose electrophoresis of concentrated HPLC fractions (left); (D) HPLC chromatogram of crude 3′-biotinylated mRNA N₃-m⁷GRNA_gluc-Biot (right) and electrophoretic mobility shift assay (EMSA) of crude and HPLC-isolated products of 3′ biotinylation (left).

Figure 4.

Application of dually labelled RNA probes for enzymatic activity monitoring. (A) Sequence and theoretical secondary structures of Cy5-RNA₃₅-Cy3, Cy5-m⁷GRNA₃₅-Cy3 and Cy5-m⁷GRNA₂₇₆-Cy3 probes and illustration of studied DNA sequences complementary to regions of the probes. (B) Time-dependent changes of emission spectra and Cy3/Cy5 fluorescence intensity ratio after addition of RNase A, RiboLock and RNase A, RNase T1, RNase R or Dcp1/2 to Cy5-RNA₃₅-Cy3 or Cy5-m⁷GRNA₃₅-Cy3 probes. (C) Time-dependent changes of emission spectra, PAGE of reaction products, and Cy3/Cy5 fluorescence intensity ratio after addition of RNase H to probe (Cy5-RNA₃₅-Cy3 or Cy5-m⁷GRNA₂₇₆-Cy3) or DNA-probe duplex.

Figure 5.

Fluorescent mRNA transfection into HeLa cells. (A) Electrophoretic resolution of HPLC-purified mRNAs encoding eGFP (N₃-m⁷GRNA_egfp, N₃-m⁷GRNA_egfp-Cy3, Cy5-m⁷GRNA_egfp and Cy5-m⁷GRNA_egfp-Cy3) or Gaussia luciferase (ARCA-RNA_gluc, ARCA-RNA_gluc-Cy3, N₃-m⁷GRNA_gluc, N₃-m⁷GRNA_gluc-mock, N₃-m⁷GRNA_gluc-Cy3, Cy5-m⁷GRNA_gluc and Cy5-m⁷GRNA_gluc-Cy3) used for HeLa transfections. (B) Relative expression of Gaussia luciferase after mRNA transfection as a function of the presence of 3′ Cy3 modification and 5′ cap structure. (C) Flow cytometry readouts after transfection of fluorescent mRNA encoding eGFP. (D) Time-lapse microscopy images of HeLa cells transfected with Cy5-m⁷GRNA_egfp-Cy3 mRNA. Time scale set for 0 h at the beginning of recording of the images (1 h after transfection start).

Figure 6.

Microinjection of zebrafish embryos with modified mRNA. (A) Schematic representation of the experimental set-up and injection of mRNA in the course of zebrafish development during the first 48 h post fertilization (hpf). (B–D) Confocal microscopy images of embryos injected with 300 pg of N₃-m⁷GRNA_egfp, N₃-m⁷GRNA_egfp-Cy3, Cy5-m⁷GRNA_egfp or Cy5-m⁷GRNA_egfp-Cy3 mRNA captured at 8 hpf (B) or 24 hpf in tail (C) or head (D) sections. Bright field and GFP fluorescence images of whole embryos are presented in Supplementary Figure S24.