Synthesis and evaluation of an alkyne-modified ATP analog for enzymatic incorporation into RNA

Abstract

Alkyne-modified nucleoside analogs are useful for nucleic acid localization as well as functional and structural studies because of their ability to participate in copper-catalyzed azide/alkyne cycloaddition (CuAAC) reactions. Here we describe the synthesis of the triphosphate of 7-ethynyl-8-aza-7-deazaadenosine (7-EAATP) and the enzymatic incorporation of 7-EAA into RNA. The free nucleoside of 7-EAA is taken up by HeLa cells and incorporated into cellular RNA by endogenous RNA polymerases. In addition, 7-EAATP is a substrate for both T7 RNA polymerase and poly (A) polymerase from Escherichia coli in vitro, albeit at lower efficiencies than with ATP. This work adds to the toolbox of nucleoside analogs useful for RNA labeling.

Keywords: ATP analogs; Cellular RNA labeling; Click chemistry; Polyadenylation; T7 transcription.

Figure 1

7-Ethynyl-8-aza-7-deazaadenosine (7-EAA) base pairs with uridine (U) in duplex RNA and projects a terminal alkyne into the major groove.

Figure 2

7-EAA is taken up by HeLa cells and incorporated into cellular RNA. A) Detection of biotin-labeled RNA after click reaction with biotin-azide (M) biotinylated molecular weight markers in kb, (1) Sample isolated from cells grown in absence of 7-EAA, treated with RNase V1; (2) Sample isolated from cells grown in the presence of 100 μM 7EAA, treated with RNase V1; (3) Sample isolated from cells grown in the absence of 7-EAA, no RNase V1 treatment; (4) Sample isolated from cells grown in presence of 100 μM 7-EAA, no RNase V1 treatment. B) Same as in A) with ethidium bromide (EtBr) detection instead of biotin detection.

Figure 3

Synthesis of 17 nt RNA by in vitro transcription with 7-EAATP and T7 RNA polymerase. A) T7 RNA polymerase transcription template for synthesis of 17 nt RNA containing one 7-EAA or A residue (X). B) Products of T7 polymerase transcriptions with template shown in (A) containing 5 mM ATP or 7-EAATP, α -³²P GTP and allowed to proceed for 2 h at 37 °C. (arrow indicates major transcription product). C) CuAAC reaction with 17 nt RNAs. Lanes 1-3: product from transcription with 7-EAATP, Lanes 4-6: product from transcription with ATP. Lanes 1 and 4: no CuAAC reaction reagents added, Lanes 2 and 5: CuAAC reagents lacking azide, Lanes 3 and 6: CuAAC reagents plus azide. (*) indicates position of CuAAC product.

Figure 4

Comparison of T7 RNA polymerase efficiency with template shown in Fig. 3A using varying concentrations of 7-EAATP or ATP.

Figure 5

Synthesis of 335 nt RNA by in vitro transcription with 7-EAATP and T7 RNA polymerase. A) Northern blot of RNA transcribed in the presence of different ratios of ATP:7-EAATP followed by click reaction modification with a biotin azide and detection for the presence of biotin. M: molecular weight markers in kb, Lane 1: no 7-EAATP in transcription, Lane 2: ATP:7-EAATP ratio is 1:10, Lane 3: ATP:7-EAATP ration is 1:5 B) Lanes are same as in A with ethidium bromide detection.

Figure 6

RNA poly(A) tailing with 7-EAATP. A) Biotin detection for RNA with poly(A) tail. B) RNA stain (SYBR® safe) detection for RNA with poly(A) tail. Lane M contains molecular marker in kb. Lanes 1 to 3 contain poly(A) tailed RNAs with different ATP:7EAATP ratios in each reaction. Lane 4 contains RNA without poly(A) tail.

Scheme 1

Synthesis of 7-EAATP. i) TMS-ethyne, Pd(PPh₃)₄, CuI, Et₃N, THF, 87%; ii) DBU, MeOH, 60%; iii) (MeO)₃PO, proton sponge, POCl₃, −15 °C; iv) (HNBu₃)₂H₂P₂O₇, NBu₃, DMF, −15 °C; v) TEAB, rt, 11% (three steps).