Direct and site-specific quantification of RNA 2'-O-methylation by PCR with an engineered DNA polymerase

Abstract

Methylation of the 2'-hydroxyl-group of ribonucleotides is found in all major classes of RNA in eukaryotes and is one of the most abundant posttranscriptional modifications of stable RNAs. In spite of intense studies, the multiple functions of RNA 2'-O-methylation are still not understood. One major obstacle in the field are the technical demanding detection methods, which are typically laborious and do not always deliver unambiguous results. We present a thermostable KlenTaq DNA polymerase variant with significant reverse transcription activity that is able to discriminate 2'-O-methylated from unmethylated RNAs. The engineered enzyme catalyzes DNA synthesis from DNA as well as RNA templates and enables expeditious quantification of 2'-O-methylation of individual nucleotides directly from total RNA extracts by a simple qRT-PCR.

Figure 1.

DNA synthesis catalyzed by RT-KTQ-LSIM is hampered by 2′-O-methylation of RNA templates. (A) Structures of relevant nucleotides. (B) Primer extension in presence of methylated or unmethylated RNA templates catalyzed by RT-KTQ-LSIM. (C) qRT-PCR amplification of methylated and unmethylated RNA oligonucleotides catalyzed by RT-KTQ-LSIM.

Figure 2.

Rational design of RT-KTQ-LSIM libraries. Amino acids in immediate proximity to the 2′-oxygen of the nucleotide paired to the incoming dNTP were selected for saturation mutagenesis (namely G668, V669, G672, R746, K747 and N750). Adapted from PDB 4BWM (24) using PyMOL (Schrödinger, LLC, New York, NY, USA).

Figure 3.

RT-KTQ-LSIM V669L features increased discrimination between 2′-O-methylated and unmethlyated RNA templates and enables quantification of 2′-O-methylation by qRT-PCR. (A) Primer extension in the presence of methylated or unmethlyated RNA templates catalyzed by RT-KTQ-LSIM V669L. (B) qRT-PCR amplification of methylated and unmethylated RNA oligonucleotides catalyzed by RT-KTQ-LSIM V669L. (C) RT-PCR reactions were stopped after 25 cycles (top) or 30 cycles (bottom) and analyzed by agarose gel electrophoresis. (D) The ΔC_T-method was used to calculate methylation ratio of RNA template at 100 pM concentration with varied fractions of 2′OmeA/A at the target position. Error bars describe SD (n = 3).

Figure 4.

Quantification of ribosomal methylation directly from total RNA by qRT-PCR. (A) Analysis of the methylation status of A27, A99, U428, G1490 and C1703 in 18s rRNA from total RNA extracts of various human cell lines. Error bars describe SD. (B) qRT-PCR data of methylation site A99 in HEK-293 and Caco2 cells. ΔC_T values indicate higher degree of methylation in HEK-293 cells than in Caco2 cells.