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

Circular single-stranded RNAs (C-RNAs) are RNA rings without terminals, which are promising for many practical applications both in vivo and in vitro. Recently, for example, thousands of circular RNAs (circRNAs) have been found almost in all kingdom of organisms, and their biological roles as miRNA or RNA inhibitors have been reported. Many circRNAs have also been found to be translated or regulate functions of other proteins. Accordingly, artificial synthesis of C-RNAs has attracted much more interest for significant applications. One of the most advantageous properties of C-RNAs, which greatly facilitates their practical applications, is that they are strongly resistant against exo-ribonucleases, compared with non-cyclic linear RNAs (L-RNAs) (16–24). For example, siRNA effects of dumbbell-shaped C-RNAs were very high in living cells, primarily because of their long life-time. By combining the ring of antisense RNA strand with linear sense strand, off-target effects were reduced. Cyclic ribozymes and cyclic RNA aptamers were also developed to regulate their functions. Rolling circle translation of C-RNAs in cells was reported (25,26). Furthermore, circularization of L-RNAs is also useful to temporarily cage their intrinsic functions (e.g. ribozyme activity and gene-silencing).
In most cases, C-RNAs are synthesized by circularizing L-RNAs with the use of enzymes. T4 RNA ligase 2 (T4 Rnl2) has been often used. The pioneering work by Yin et al. showed that some L-RNAs were circularized by this enzyme to the rings. However, direct applications of this finding to preparation of various C-RNAs are not very successful, since the efficiency of circularization is highly dependent on the structure of L-RNA. Accordingly, in most cases, auxiliary oligonucleotide (called as splint), which is complementary with the terminal portions of L-RNA, was added to place the two ends close to each other. The resultant nick structure is recognized by Rnl2 (a dsRNA ligase). A similar methodology is being employed to prepare the rings of single-stranded DNA. It has been found that appropriate efficiency is favourable to suppress intermolecular ligation of ssDNA circularization. With this method, however, desired intramolecular RNA circularization is always accompanied by troublesome intermolecular polymerization of L-RNA, especially for higher RNA concentrations (Figure ​(Figure1B).1B). This concurrent polymerization notably lowers the yields of C-RNAs, and complicates the synthetic procedures. Furthermore, smaller-sized rings are hardly obtainable with this strategy, since cyclic intermediates involving short L-RNA (e.g. <30 nt) and splint are sterically unstable. Even with the use of T4 RNA ligase 1 (a single-stranded RNA specific ligase), intermolecular polymerization is dominant at high-substrate concentrations in many cases. In order to reduce the polymerization, specially designed DNA templates (helper) must be used. Circularization by ssRNA circligase™ (Epicentre Co.) requires Mn2+ as an essential cofactor, and relatively large amount of ligase is essential for high yield of ligation even at a low concentration of substrates. 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.