Preferential production of RNA rings by T4 RNA ligase 2 without any splint through rational design of precursor strand

Rings of single-stranded RNA are promising for many practical applications, but the methods to prepare them in preparative scale have never been established. Previously, RNA circularization was achieved by T4 RNA ligase 2 (Rnl2, a dsRNA ligase) using splints, but the yield was low due to concurrent intermolecular polymerization. Here, various functional RNAs (siRNA, miRNA, ribozyme, etc.) are dominantly converted by Rnl2 to the rings without significant limitations in sizes and sequences. The key is to design a precursor RNA, which is highly activated for the efficient circularization without any splint. First, secondary structure of target RNA ring is simulated by Mfold, and then hypothetically cut at one site so that a few intramolecular base pairs are formed at the terminal. Simply by treating this RNA with Rnl2, the target ring was selectively and efficiently produced. Unexpectedly, circular RNA can be obtained in high yield (>90%), even when only 2 bp form in the 3′-OH side and no full match base pair forms in the 5′-phosphate side. Formation of polymeric by-products was further suppressed by diluting conventional Rnl2 buffer to abnormally low concentrations. Even at high-RNA concentrations (e.g. 50 μM), enormously high selectivity (>95%) was accomplished.

A streamlined protocol for the detection of mRNA–sRNA interactions using AMT-crosslinking in vitro

Until recently, RNA–RNA interactions were mainly identified by crosslinking RNAs with interacting proteins, RNA proximity ligation and deep sequencing. Recently, AMT-based direct RNA crosslinking was established. Yet, several steps of these procedures are rather inefficient, reducing the output of identified interaction partners. To increase the local concentration of RNA ends, interacting RNAs are often fragmented. However, the resulting 2′,3′-cyclic phosphate and 5′-OH ends are not accepted by T4 RNA ligase and have to be converted to 3′-OH and 5′-phosphate ends. Using an artificial mRNA/sRNA pair, we optimized the workflow downstream of the crosslinking reaction in vitro. The use of a tRNA ligase allows direct fusion of 2′,3′-cyclic phosphate and 5′-OH RNA ends.

Synthesis of RNA Bearing C5-polyamine Modified Pyrimidine Nucleosides at the 3´-end by T4 RNA Ligase

The incorporation of the modified nucleoside into the 3´-overhang regions of short interfering RNAs (siRNAs) has been reported to enhance their nuclease resistance and improve RNA interference activity. In this study, DNA 2-mers containing C5-polyamine-modified pyrimidine nucleosides were synthesized and then ligated to the 3´-end of the RNA by T4 RNA ligase. The modification of the base moiety with tris(2-aminoethyl)amine affected the ligation efficiency, but the DNA 2-mer containing only one modified nucleoside was ligated with sufficient efficiency. We propose a novel synthetic route to modified siRNA bearing various modified groups in the 3´-overhang region.

RNA ligation of very small pseudo nick structures by T4 RNA ligase 2, leading to efficient production of versatile RNA rings

T4 Rnl2 ligates ssRNA via nick-like structures, leading to efficient production of versatile RNA rings for various applications.

Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD+-dependent polynucleotide ligases

Polynucleotide ligases comprise a ubiquitous superfamily of nucleic acid repair enzymes that join 3′-OH and 5′-PO4DNA or RNA ends. Ligases react with ATP or NAD+and a divalent cation cofactor to form a covalent enzyme-(lysine-Nζ)–adenylate intermediate. Here, we report crystal structures of the founding members of the ATP-dependent RNA ligase family (T4 RNA ligase 1; Rnl1) and the NAD+-dependent DNA ligase family (Escherichia coliLigA), captured as their respective Michaelis complexes, which illuminate distinctive catalytic mechanisms of the lysine adenylylation reaction. The 2.2-Å Rnl1•ATP•(Mg2+)2structure highlights a two-metal mechanism, whereby: a ligase-bound “catalytic” Mg2+(H2O)5coordination complex lowers the pKaof the lysine nucleophile and stabilizes the transition state of the ATP α phosphate; a second octahedral Mg2+coordination complex bridges the β and γ phosphates; and protein elements unique to Rnl1 engage the γ phosphate and associated metal complex and orient the pyrophosphate leaving group for in-line catalysis. By contrast, the 1.55-Å LigA•NAD+•Mg2+structure reveals a one-metal mechanism in which a ligase-bound Mg2+(H2O)5complex lowers the lysine pKaand engages the NAD+α phosphate, but the β phosphate and the nicotinamide nucleoside of the nicotinamide mononucleotide (NMN) leaving group are oriented solely via atomic interactions with protein elements that are unique to the LigA clade. The two-metal versus one-metal dichotomy demarcates a branchpoint in ligase evolution and favors LigA as an antibacterial drug target.