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Modified RNA plays a key role in many biochemical and biophysical techniques for the investigation of RNA structures and function and for use in cutting edge technologies as an aptamer or siRNA. Among several positions for site-specific labeling of RNA, the 20-position of the sugar moiety is of major interest, since modifications can be incorporated in both purine and pyrimidine nucleosides. 20-Modified RNA is used for technologies in which increased nuclease resistance and chemical stability are of great importance and in which naturally occurring nucleic acids have limitations. Furthermore, the 20-ribose position has been shown to be advantageous for the introduction of selenium into RNA for use in X-ray crystallography. In addition to commonly used chemical solid phase synthesis of modified RNA using phosphoramidite building blocks, the enzymatic synthesis of modified RNA using T7 RNA polymerase and modified ribonucleoside 50-triphosphates (NTPs) has been more and more investigated over the past few years. The demand for T7 RNA polymerase mutants generated by protein engineering that are able to incorporate modified nucleotides more efficiently therefore raises the need for new screening systems for selection of interesting candidates from mutant libraries. Directed protein evolution as an iterative process of mutagenesis, expression and screening or selection has been successfully used to establish DNA polymerases with altered properties like the ability to incorporate ribonucleotides, modified nucleotides like 20-O-methyl ribonucleotides or with increased reverse transcriptase activity for a variety of applications. Several reported selection methods like phage display or compartmentalized self-replication have been developed and different screening platforms based on fluorescence readout of synthesized DNA have been established to evolve DNA polymerase mutants with altered properties. Regarding the expansion of the substrate spectra of RNA polymerases, only a few approaches based on rational design and site-directed mutagenesis of T7 RNA polymerase have been reported so far. Recently, the directed evolution of a T7 RNA polymerase mutant, referred to as ‘RGVG’ E593G, V685A, with increased acceptance for 20-O-methyl NTPs (20-OMe-NTPs) was reported. Previously, we have investigated the applicability of two T7 RNA polymerase mutants for the enzymatic synthesis of 20-methylseleno (20-SeMe) modified RNA. We demonstrated the efficient enzymatic synthesis of 20-SeMe-modified RNA using mutant ‘RGVG’ E593G, V685A and thus provided a foundation for an alternative derivatization strategy for RNA structure determination using the multiwavelength anomalous dispersion technique (MAD). Encouraged by our previous findings, we here report the screening of T7 RNA polymerase mutants with increased acceptance of 20-SeMe-UTP and 20-OMe-UTP. We started with the construction of a T7 RNA polymerase mutant library by randomizing amino acid positions Y639 and H784. Both residues are located in the active site of the polymerase and have been shown to be involved in ribose recognition events and nucleotide discrimination. DNA fragments containing each of the randomized regions were prepared by PCR using oligonucleotide primers in which the codons of amino acids Y639 and H784 were randomized. The fragments were purified and assembled in a second step by overlap extension PCR. Afterwards, the assembled PCR product was cloned into a vector (pGDR11) suitable for protein expression in E. coli BL21 cells under control of an IPTG (isopropyl-b-D-thiogalactopyranoside) inducible T5 promotor. Electrocompetent E. coli BL21 cells were transformed with the mutant library and codon randomization was verified by sequencing the T7 RNA polymerase gene of several transformants. We picked 3200 transformants and cultivated them in 384-deepwell plates for further storage at 80 1C. The mutants from the library were expressed, E. coli cells harvested and lysed using lysozyme in 96-well plates as previously described for the screening of DNA polymerases. Cell lysates containing the overexpressed T7 RNA polymerase mutants were diluted with buffer and directly used for activity screening. Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany. E-mail: [email protected]; Fax: +49 7531-885140; Tel: +49 7531-885139 b Institute of Organic Chemistry, Center for Chemistry and Biomedicine, Leopold-Franzens University, Innrain 80-82, 6020 Innsbruck, Austria w Electronic supplementary information (ESI) available. See DOI: 10.1039/c2cc35028a ChemComm Dynamic Article Links