RNA ligation joins compatible RNA termini by forming a new phosphodiester bond. Joining two fragments extends the connected chain; joining the two ends of one fragment produces circular RNA. The end chemistry matters: an ATP-dependent RNA ligase can join a hydroxyl group to a phosphate, whereas RtcB uses a different combination of terminal groups.[ref][ref]
An unmodified linear four-nucleotide fragment has three backbone links, and a six-nucleotide fragment has five. One intermolecular ligation gives ten nucleotides connected by links in a linear chain. Circularizing that chain adds one more backbone link while retaining ten nucleotides. Counting chain units, connecting bonds and separate molecules therefore answers three different questions.
A 1972 experiment made end joining measurable using radioactively labelled RNA. After reaction with a purified RNA ligase, labelled terminal phosphate became resistant to alkaline phosphatase. Additional tests with enzymes attacking from chain ends supported formation of circular poly(A) under the studied conditions.[ref]
Purification and properties of bacteriophage T4-induced RNA ligase by R. Silber and others. Abstract, phosphate-label protection and exonuclease tests
A single protected label would show a chemical change at that end; the complementary end-accessibility tests made the circular-product interpretation stronger. New to topics? Read the docs here!