A versatile toolbox for posttranscriptional chemical labeling and imaging of RNA

Abstract

Cellular RNA labeling strategies based on bioorthogonal chemical reactions are much less developed in comparison to glycan, protein and DNA due to its inherent instability and lack of effective methods to introduce bioorthogonal reactive functionalities (e.g. azide) into RNA. Here we report the development of a simple and modular posttranscriptional chemical labeling and imaging technique for RNA by using a novel toolbox comprised of azide-modified UTP analogs. These analogs facilitate the enzymatic incorporation of azide groups into RNA, which can be posttranscriptionally labeled with a variety of probes by click and Staudinger reactions. Importantly, we show for the first time the specific incorporation of azide groups into cellular RNA by endogenous RNA polymerases, which enabled the imaging of newly transcribing RNA in fixed and in live cells by click reactions. This labeling method is practical and provides a new platform to study RNA in vitro and in cells.

Figure 1.

Schematic diagram illustrating the posttranscriptional chemical labeling of RNA transcripts in vitro and in cells by using azide-modified UTP analogs.

Scheme 1.

General scheme for the synthesis of azide-modified UTP analogs 4–6 from ribonucleosides 1–3, respectively (Supplementary Data).

Figure 2.

Incorporation of azide-modified UTP analogs (4–6) into RNA ONs by in vitro transcription reactions. Incorporation of 4–6 in the presence of DNA template T1 will produce RNA transcripts 7–9, respectively. Transcripts resulting from T2–T5 are also shown.

Figure 3.

Incorporation of modified UTPs into RNA ONs by T7 RNA polymerase. Transcription reactions were performed with templates T1–T5 in the presence of UTP and or modified UTPs 4 and 6. Relative% incorporation of azide-modified UTPs into modified full-length transcript is given with respect to the amount of full-length transcript formed in the presence of natural NTPs. For complete gel image see Supplementary Figure S1. Gel image of transcription reactions with UTP 5 is also provided in Supplementary Figure S1 (43).

Figure 4.

Building blocks for posttranscriptional chemical modification (Supplementary Data).

Figure 5.

Posttranscriptional chemical functionalization of azide-modified RNA ONs 7–9 by CuAAC, SPAAC and Staudinger ligation reactions. For chemical structure of the products see Supplementary Scheme S4–S6.

Figure 6.

Imaging cellular RNA transcription using AMUTP 4. Cultured HeLa cells were transfected with 4 (50 μM to 1 mM) for 1 h using DOTAP. The cells were then fixed, permeabilized and the AMU-labeling was detected by performing CuAAC reaction with Alexa594-alkyne 11.

Figure 7.

(A) AMUTP 4 is incorporated into RNA and not into DNA. HeLa cells were incubated with 15 mM hydroxyurea (ribonucleotide reductase inhibitor) and EdU/4, and were click-stained as before using Alexa594-azide (17) or Alexa594-alkyne (11). DOTAP was used when cells were incubated with 4. Inhibition of DNA synthesis by using hydroxyurea abolished the incorporation of EdU. No significant change in staining pattern in cells incubated with 4 in the presence of hydroxyurea indicates that 4 is not incorporated into DNA. (B) AMUTP 4 specifically labels transcribing RNA in cells. HeLa cells were transfected with 4 in the presence and absence of variable concentrations of actinomycin D, an RNA polymerase inhibitor. Progressive reduction in fluorescence signal in cells treated with actinomycin D confirms specific AMU labeling of cellular RNA.

Figure 8.

Imaging cellular RNA transcription using AMUTP 4 under copper-free SPAAC reaction conditions. HeLa cells were transfected with 1 mM of AMUTP 4 for 1 h. The cells were fixed and stained by SPAAC reaction using Cy3-cyclooctyne substrate 14 (red). In a parallel experiment, cells transfected with AMUTP were stained by first SPAAC reaction using 14 (red) followed by CuAAC reaction using Alexa488-alkyne 16 (green). Bottom panel is a merge of all three channels (blue, red and green). Boxed cell has been magnified.

Figure 9.

Simultaneous visualization of DNA and RNA synthesis in cells by CuAAC reactions. In separate experiments, HeLa cells were incubated with EdU/AMUTP and a combination of EdU and AMUTP. While EdU labeling in DNA was detected by a click reaction with Alexa594-azide 17 (red), AMU labeling in RNA was detected by a reaction with Alexa488-alkyne 16 (green). Bottom panel is a merge of all three channels (blue, red and green). Boxed cell has been magnified.