Expanding the chemical scope of RNA:methyltransferases to site-specific alkynylation of RNA for click labeling

Abstract

This work identifies the combination of enzymatic transfer and click labeling as an efficient method for the site-specific tagging of RNA molecules for biophysical studies. A double-activated analog of the ubiquitous co-substrate S-adenosyl-l-methionine was employed to enzymatically transfer a five carbon chain containing a terminal alkynyl moiety onto RNA. The tRNA:methyltransferase Trm1 transferred the extended alkynyl moiety to its natural target, the N2 of guanosine 26 in tRNA(Phe). LC/MS and LC/MS/MS techniques were used to detect and characterize the modified nucleoside as well as its cycloaddition product with a fluorescent azide. The latter resulted from a labeling reaction via Cu(I)-catalyzed azide-alkyne 1,3-cycloaddition click chemistry, producing site-specifically labeled RNA whose suitability for single molecule fluorescence experiments was verified in fluorescence correlation spectroscopy experiments.

Figure 1.

(A) S-Adenosyl-l-methionine (AdoMet or SAM, 1) and the substrate analog AdoEnYn 2. (B) Enzymatic transfer of tritiated methyl groups from [methyl-³H]AdoMet to RNA by the Trm1 enzyme is inhibited by 2. (C) Eadie-Hofstee plot of Trm1 inhibition by AdoEnYn 2. Activity of Trm1 was tested as described in ‘Materials and Methods’ section, except that AdoMet was used at variable concentrations ranging from 0.25 to 8.0 µM. AdoEnYn 2 was added at concentrations of 10, 50 and 250 µM, as indicated in the graph. Data are plotted in the V versus V/[S] coordinates where V denotes the speed of tRNA methylation expressed in pmol/min and [S] the substrate concentration in micromolar.

Figure 2.

LC/UV (254 nm) and LC/MS (m/z 348) analysis of modified tRNA^Phe transcript after enzymatic digestion shows the formation of alkynylated guanosine 3; an MS taken from the chromatogram at 17.4 min is shown in the lower left. LC/MS/MS shows the typical fragmentation of 3 (m/z 348) to the alkynylated nucleobase 4 (m/z 216; lower right).

Figure 3.

Click derivatization of EnYn-modified RNA by a fluorescent azide. The reaction scheme is shown in (A). Reactions were analyzed by denaturing PAGE and subsequent scanning for AlexaFluor 594 fluorescence (Excitation 532 nm, Emission 610 nm; upper left). A loading control was obtained after RNA staining with SYBRGold (B).

Figure 4.

Identificaton of the fluorescent adduct of AlexaFluor 594 azide and the modified guanosine 3. Panels on the left side show HPLC with DAD detection of nucleosides (A) and Alexa dye (B), and fluorescence detection (C). Panels on the right side show detection on an LC/MS/MS setup by UV absorption (D) and tracing of the [M + H]²⁺ mother ion (E). Panel (F) shows an MS of the peak eluting at 20.5 min (note that the given structural formulas contain a positive charge and single protonation will lead to an ion with m/z = 2). LC/MS/MS fragmentation of the mother ion yielded a strong signal resulting from loss of the ribose moiety (G). Note that, due to altered capillary lengths between the various detectors, the peaks show slightly different retention in the DAD/FLD setup (A–C) than in the LC/MS/MS setup (D–G).

Figure 5.

RT arrest by the enzymatically introduced EnYn side chain followed by AlexaFluor 594 modification in tRNA^Phe from yeast. A PAGE analysis of the RT arrest assay with untreated and labeled tRNA^Phe is compared to sequencing reactions. Modification of G₂₆ caused an arrest of primer elongation at nt 27, as indicated in the cloverleaf structure on the right. Nucleotide numbering is according to the Sprinzl database (49).

Figure 6.

Concentration dependance for EnYn transfer to tRNA^Phe transcript by Trm1. The AdoEnYn 2 concentration was varied from 2 to 64 µM in the kinetic assay. EnYn-modified tRNA^Phe was then EtOH precipitaed and subjected to the CuAAC click reaction. Small molecules were removed from the Alexa 594-labeled tRNA^Phe using G-25 spin columns and the RNA analyzed by 10% urea PAGE followed by SYBRGold staining. Fluorescence scanning and quantification was done for both fluorophores (A) and the ratio of Alexa 594/SYBRGold fluorescence is plotted against the AdoEnYn 2 concentration (B).

Figure 7.

FCS of AlexaFluor 594-labeled tRNA^Phe transcript. Normalized FCS autocorrelation curves (symbols) and the corresponding fits (lines) for free AlexaFluor 594 (black circles) and AlexaFluor594-labeled tRNA^Phe (red squares).

Figure 8.

Exploratory screen with four different tRNA:MTases. The reaction with AdoEnYn 2 was performed at standard conditions using variable amounts of Trm1, Trm4, TrmI and Trm11 (see ‘Materials and Methods’ section). Analysis was done as described before. The composite panel shows the loading control in the lower part. Large bands appearing in white result from xylene cyanol dye present in the loading buffer.