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.

Is DNA or RNA a good nutrient? Although scientists have raised this question for dozens of years, few textbooks mention the nutritional role of nucleic acids. Paradoxically, mononucleotides are widely added to infant formula milk and animal feed. Interestingly, competent bacteria can bind and ingest extracellular DNA and even integrate it into their genome. These results prompt us to clarify whether bacteria can “eat” DNA as food. We found that Escherichia coli can grow well in the medium with DNA as carbon and nitrogen sources. More interestingly, in the presence of glucose and DNA, bacteria grew more rapidly, showing that bacteria can use DNA as an excellent nitrogen source. Surprisingly, the amount of DNA in the culture media decreased but its length remained unchanged, demonstrating that E. coli ingested long DNA directly. The gene expression study shows that E. coli mainly ingests DNA before digestion and digests it in the periplasm. Bifidobacterium bifidum can also use DNA as the nitrogen source for growth, but not efficiently as E. coli. This study is of great significance to study DNA metabolism and utilization in organisms. It also lays a foundation to understand the nutritional function of DNA in intestinal flora and human health.

Accelerated aggregation of DNA-functionalized gold nanoparticles is discovered in alcoholic solvents upon adding full-match DNA and is potentially useful for the identification of Baijiu.

When oligonucleotide bearing a hairpin near either its 3'- or 5'-end was treated with T4 DNA ligase, the intramolecular cyclization dominantly proceeded and its monomeric cyclic ring was obtained in extremely high selectivity. The selectivity was hardly dependent on the concentration of the oligonucleotide, and thus it could be added in one portion to the mixture at the beginning of the reaction. Without the hairpin, however, the formation of polymeric byproducts was dominant under the same conditions. Hairpin-bearing oligonucleotides primarily take the folded form, and the enzymatically reactive species (its open form) is minimal. As the result, the intermolecular reactions are efficiently suppressed due to both thermodynamic and kinetic factors. The 'terminal hairpin strategy' was applicable to large-scale preparation of a variety of DNA rings. The combination of this methodology with 'diluted buffer strategy', developed previously, is still more effective for the purpose. When large amount of l-DNA bearing a terminal hairpin (e.g. 40 μM) was treated in a diluted ligase buffer (0.1× buffer) with T4 DNA ligase, the DNA ring was prepared in 100% selectivity. Even at [l-DNA]0 = 100 μM in 0.1× buffer, the DNA ring was also obtained in pure form, simply by removing tiny quantity of linear byproducts by Exonuclease I.