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Abstract Innovative approaches to controlled nucleobase-modified RNA synthesis are urgently needed to support RNA biology exploration and to synthesize potential RNA therapeutics. Here we present a strategy for enzymatic construction of nucleobase-modified RNA based on primer-dependent engineered thermophilic DNA polymerases – SFM4-3 and TGK. We demonstrate introduction of one or several different base-modified nucleotides in one strand including hypermodified RNA containing all four modified nucleotides bearing four different substituents, as well as strategy for primer segment removal. We also show facile site-specific or segmented introduction of fluorophores or other functional groups at defined positions in variety of RNA molecules, including structured or long mRNA. Intriguing translation efficacy of single-site modified mRNAs underscores the necessity to study isolated modifications placed at designer positions to disentangle their biological effects and enable development of improved mRNA therapeutics. Our toolbox paves the way for more precise dissecting RNA structures and functions, as well as for construction of diverse types of base-functionalized RNA for therapeutic applications and diagnostics.

Directionality of auxin transport is determined by polar localization of PIN-FORMED (PIN) proteins. Here the authors show that Arabidopsis WAV3 E3 ubiquitin ligases contribute to PIN polarity by selectively antagonizing ectopic deposition of de novo synthesized PIN proteins following cell division.

Immune tolerance is mediated by the deletion of autoreactive T cells via medullary thymic epithelial cells (mTEC) and dendritic cells (DC), and by the induction of regulatory T cells (Treg). Here the authors show that mTEC receiving toll-like receptor signaling control the recruitment of CD14+Sirpα+ DC population that is capable of inducing Treg for establishing tolerance.