A dual-purpose polymerase engineered for direct sequencing of pseudouridine and queuosine RNA modifications

More than 170 posttranscriptional RNA modifications are so far known on both coding and noncoding RNA species. Within this group, pseudouridine (Ψ) and queuosine (Q) represent conserved RNA modifications with fundamental functional roles in regulating translation. Current detection methods of these modifications, which both are reverse transcription (RT)-silent, are mostly based on the chemical treatment of RNA prior to analysis.

To overcome the drawbacks associated with indirect detection strategies, University of Konstanz researchers have engineered an RT-active DNA polymerase variant called RT-KTq I614Y that produces error RT signatures specific for Ψ or Q without prior chemical treatment of the RNA samples. Combining this polymerase with next-generation sequencing techniques allows the direct identification of Ψ and Q sites of untreated RNA samples using a single enzymatic tool.

Strategy for the detection of the RNA modifications Ψ and Q by increased misincorporation

Strategy for the detection of the RNA modifications Ψ and Q by increased misincorporation. During the RT step, the DNA polymerase enzyme incorporates higher levels of mismatched nucleotides opposite modified nucleotides (right side, mismatch depicted as circle on the cDNA). Resulting errors within the cDNA strand are retained during the following amplification steps and detected by NGS. Sites of higher error rates (error hot spots) are used to distinguish modified (right) from unmodified positions (left).

During the RT step, the DNA polymerase enzyme incorporates higher levels of mismatched nucleotides opposite modified nucleotides (right side, mismatch depicted as circle on the cDNA). Resulting errors within the cDNA strand are retained during the following amplification steps and detected by NGS. Sites of higher error rates (error hot spots) are used to distinguish modified (right) from unmodified positions (left).

Huber LB, Kaur N, Henkel M, Marchand V, Motorin Y, Ehrenhofer-Murray AE, Marx A. (2023) A dual-purpose polymerase engineered for direct sequencing of pseudouridine and queuosine. Nucleic Acids Res [Epub ahead of print]. [article]

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