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Ribonuclease T1 is a guanosine-selective ribonuclease: it cleaves susceptible phosphodiester bonds on the 3′ side of guanosine residues in single-stranded RNA. Its two-stage chemistry first creates an RNA cyclic phosphate end and a 5′-hydroxyl end, then converts the cyclic end to a 3′-phosphate end by hydrolysis.^[ref]

The current classification is EC 4.6.1.24, a lyase entry. The former EC 3.1.27.3 entry was transferred in 2020 because the primary cleavage step is a phosphorus–oxygen lyase reaction. Merely describing the final overall reaction as hydrolysis hides that first step.^[ref]

An invented sequence example makes its selectivity concrete. Read ACGUAAGCU from 5′ to 3′ and suppose all eligible internal bonds are accessible and completely cleaved. Cutting after each G gives ACG|UAAG|CU: fragments of three, four and two nucleotides, respectively. The nine nucleotides are redistributed among three fragments rather than disappearing. For a linear chain, $k$ distinct internal cuts produce $k+1$ fragments.

The letters alone do not guarantee that a folded RNA exposes every candidate bond. Sequence preference and physical accessibility jointly affect a cleavage pattern; the toy calculation explicitly assumes complete accessibility.

This selectivity also made ribonuclease T1 useful in historical sequence comparisons. A 1977 study characterized 16S ribosomal RNA from ten methane-producing microbial species through the fragments produced by T1 digestion, then compared the resulting catalogs.^[ref] Selective cleavage converted a large molecule into a collection of experimentally comparable sequence pieces.

An RNA cyclic phosphate end is an RNA terminus in which a phosphate group links the $2^{\prime }$ and $3^{\prime }$ oxygens of the terminal ribose, forming a small cyclic phosphodiester bond arrangement. The ring is local to the terminal nucleotide; the whole RNA chain need not be circular. This end chemistry differs from both a free $3^{\prime }$ hydroxyl group and an ordinary terminal $3^{\prime }$ phosphate.^[ref]

The difference matters for RNA ligation. A 2012 study showed that RtcB opens the cyclic end to a $3^{\prime }$ phosphate before joining it to an RNA $5^{\prime }$ hydroxyl group. The reactions depended on GTP and enzyme guanylylation. Researchers followed labelled products over time using denaturing polyacrylamide gel electrophoresis, distinguishing starting cyclic ends, transient products and joined RNA.^[ref]

As a measurement example, two four-nucleotide RNA molecules could both spell AUGC while one ends in a free $3^{\prime }$ hydroxyl group and the other in a cyclic phosphate. A readout reporting only their letters would merge them, even though a reaction requiring the free hydroxyl group could distinguish them. Nucleotide sequence therefore does not completely specify terminal chemistry, and terminal chemistry can determine which RNA molecules an assay records.

An RNA ligase is an enzyme that catalyzes covalent joining of RNA ends. Its usefulness depends on the terminal groups it recognizes and the chemistry that activates the joining reaction. The activity classified as EC6.5.1.3, an ATP-dependent RNA ligase, joins $3^{\prime }$ hydroxyl group and $5^{\prime }$ phosphate termini while converting ATP to adenosine monophosphate and diphosphate.^[ref]

RtcB illustrates another route. It joins a $3^{\prime }$ phosphate to a $5^{\prime }$ hydroxyl group using GTP, and can process an RNA cyclic phosphate end before sealing. The substrate ends appropriate for one RNA ligase therefore need not be appropriate for another.^[ref]

For a specified assay, imagine two RNA fragments held in the correct alignment. One pair has the terminal groups accepted by the chosen RNA ligase; an otherwise sequence-identical pair carries a blocked end. A failure to join the second pair could reflect end chemistry even though the letters and alignment match. A meaningful comparison separates recognition, activation and final phosphodiester bond formation. Historically, purification of a T4-induced enzyme and tests of its labelled products connected the joining activity to a biochemical preparation.^[ref] That functional evidence goes beyond assigning an enzyme name from sequence resemblance alone.

RNA repair restores damaged RNA chemistry or chain continuity. For a strand break, repair can require preparing compatible termini and then using an RNA ligase to form a new phosphodiester bond. The chemical state of an end is therefore part of the substrate: an RNA cyclic phosphate end and a free $3^{\prime }$ hydroxyl group need not enter the same joining pathway.^[ref]

In 2011, purified Escherichia coli RtcB joined a broken transfer RNA-like stem-loop with cyclic-phosphate and $5^{\prime }$ hydroxyl termini. Radioactive labelling and denaturing polyacrylamide gel electrophoresis distinguished starting fragments from joined product.^[ref] This established biochemical joining activity and motivated a cellular repair hypothesis. The assay alone did not establish every physiological substrate or prove recovery of all biological functions.

For a chain-inventory example, cut an eight-nucleotide RNA into fragments containing three and five nucleotides. Correctly reconnecting their original ends restores eight units in one chain. If one nucleotide has instead been lost, joining the remaining pieces produces seven units; ligation alone does not recreate missing sequence information. Product length, junction chemistry, nucleotide sequence and biological activity consequently provide different tests of what has been restored.

RtcB is an RNA ligase that seals an RNA $3^{\prime }$ phosphate to a $5^{\prime }$ hydroxyl group. It also accepts an RNA cyclic phosphate end, which is first opened to a linear $3^{\prime }$ phosphate. Its reaction class, EC6.5.1.8, uses GTP and a divalent metal cofactor; guanosine monophosphate and diphosphate appear in the overall accounting.^[ref]

A 2012 study separated the reaction into experimentally testable steps. GTP first supplies a guanylate group that becomes covalently attached to the enzyme. Transfer to the RNA end creates an activated intermediate; the incoming $5^{\prime }$ hydroxyl group then completes the junction. The authors followed labelled substrates and products using denaturing polyacrylamide gel electrophoresis and found that cyclic-phosphate opening was fast enough to precede joining.^[ref]

A staged interpretation explains a useful experimental distinction. A variant might open a cyclic end yet fail to complete RNA ligation; another defect might prevent the initial enzyme activation. Both could yield little final product, while accumulating different intermediates. Measuring only joined RNA would merge these failures. Resolving the intermediate species helps locate which chemical step has changed.