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A base pair is an association of two nucleobases through hydrogen bonds in a compatible arrangement. It commonly joins opposite strands of a nucleic acid double helix, but can also join two regions of one folded RNA strand. Canonical Watson–Crick DNA base pairs match adenine with thymine and guanine with cytosine; canonical RNA base pairs use adenine with uracil instead. The nucleobases belong to complete nucleotides, including their attached sugars and backbone phosphate. See NHGRI: base pair.

The abbreviation bp also measures the length of a paired region. Our ACGTAC/TGCATG duplex has six base pairs, containing twelve nucleotides altogether. Its upper strand contains two adenine, two cytosine, one guanine, and one thymine nucleobase; the lower contains one, one, two, and two respectively. Combined, the counts are three of each nucleobase. Equality of total adenine and thymine, or of total guanine and cytosine, need not hold within either individual strand.

Base pairing is the recognition and association of compatible nucleobases. In canonical Watson–Crick geometry, an adenine–thymine base pair has two hydrogen bonds, while a guanine–cytosine base pair has three. These counts refer to those specific geometries and usual chemical forms; they are not a definition of every possible base pair. RNA can also contain other pairings, and canonical adenine–uracil pairing replaces adenine–thymine pairing. See Clark, Douglas and Choi, Biology 2e: DNA structure.

For this illustrative duplex, the six base pairs are A–T, C–G, G–C, T–A, A–T, and C–G. Thus three contribute two hydrogen bonds each and three contribute three each:Each dotted connector in the diagram counts one hydrogen bond; coloured boxes denote whole nucleobases, not individual chemical atoms. The solid rails denote the covalent bond scaffold. Separating the two strands disrupts base pairing and base stacking without requiring the backbone to be cut.

The count of fifteen hydrogen bonds is not fifteen identical contributions to a duplex's Gibbs free energy. Base stacking, interactions with water and ions, temperature, and the order of neighbouring base pairs also matter. Knowing the fraction of guanine–cytosine base pairs alone does not fix a nucleic acid double helix's shape or stability. See Brown, Genomes: pairing and stacking interactions. What makes base pairing especially useful is its selectivity: an exposed DNA strand constrains the partner's nucleic acid sequence, giving DNA replication a chemical recognition rule.

A nucleic acid sequence records the ordered nucleobases of a nucleic acid strand, conventionally read from $5^{\prime }$ to $3^{\prime }$. The standard DNA alphabet is adenine A, cytosine C, guanine G, and thymine T; the standard RNA alphabet uses uracil U in place of thymine T. Each letter denotes the nucleobase attached to one nucleotide, so the six-letter DNA nucleic acid sequence $5^{\prime }\text{-}\mathrm{ACGTAC}\text{-}{3}^{\prime }$ describes six linked nucleotides. See NHGRI: representing DNA by a sequence of letters.

Order carries information that composition loses. Both ACGTAC and AACCGT contain two adenine, two cytosine, one guanine, and one thymine nucleobase, but their nucleic acid sequences differ at positions 2, 3, 4, 5, and 6. At position 2, for example, the first has cytosine and the second adenine. Reporting only the four counts cannot reconstruct either ordered record.

To obtain the partner of $5^{\prime }\text{-}\mathrm{ACGTAC}\text{-}{3}^{\prime }$, apply the DNA base pairing rules position by position: A selects T, C selects G, G selects C, T selects A, A selects T, and C selects G. Alignment gives $3^{\prime }\text{-}\mathrm{TGCATG}\text{-}{5}^{\prime }$. Its opposite end labels matter because the two are antiparallel nucleic acid strands. Reading the partner from its own $5^{\prime }$ end reverses the aligned lettering and yields the reverse complement, $5^{\prime }\text{-}\mathrm{GTACGT}\text{-}{3}^{\prime }$.

Reversal alone gives CATGCA; complementation alone gives aligned TGCATG. Neither operation alone gives the partner written in the usual $5^{\prime }$-to-$3^{\prime }$ convention. Applying the reverse complement operation twice returns ACGTAC: GTACGT complements to CATGCA, which reverses to ACGTAC. This convention makes a single reported strand sufficient to specify an ideally complementary DNA duplex, provided its direction is known.

Ribonucleic acid, or RNA, is a nucleotide polymer whose nucleotide sequence can both carry information and organize a working molecular shape. Its backbone contains alternating ribose sugars and phosphate groups connected through covalent bonds. Its usual nucleobases are adenine A, uracil U, guanine G, and cytosine C. Thus RNA uses U where DNA uses T; its ribose also differs chemically from DNA's deoxyribose. See NHGRI: RNA.

In transcription, an enzyme uses one DNA strand as a template for RNA. For example, the DNA template $3^{\prime }\text{-}\mathrm{TACGG}\text{-}{5}^{\prime }$ specifies $5^{\prime }\text{-}\mathrm{AUGCC}\text{-}{3}^{\prime }$ in RNA. The base pairing rules now include A–U rather than A–T, while G–C remains. This RNA matches the other DNA strand's $5^{\prime }$-to-$3^{\prime }$ lettering after T is replaced by U. Identifying the template strand matters: replacing T by U in an arbitrarily chosen DNA strand is not a general method for deriving the RNA product. See Alberts et al., Molecular Biology of the Cell: from DNA to RNA.

RNA is often a single strand, but single-stranded does not mean straight or entirely unpaired. Complementary regions within one RNA molecule can undergo base pairing, bringing distant positions together. Consider the illustrative twelve-nucleotide nucleotide sequence $5^{\prime }\text{-}\mathrm{GCAUUUUUAUGC}\text{-}{3}^{\prime }$. Pair positions 1 with 12, 2 with 11, 3 with 10, and 4 with 9. The resulting stem-loop has four base pairs: G–C, C–G, A–U, U–A. Positions 5–8 remain unpaired in the loop. Both arms belong to the same continuous backbone, and the paired arms have the directions of antiparallel nucleic acid strands.

The diagram proposes a possible pairing pattern, not a prediction that this short RNA must adopt that shape under every condition. Actual RNA folding depends on additional interactions, temperature, ions, and associated proteins; the full three-dimensional arrangement contains more information than the drawn base pairs. The example's purpose is to show how a linear nucleotide sequence can specify contacts within itself.

Different RNA roles make this flexibility useful. Messenger RNA carries a message that a ribosome reads during translation to build a protein. Transfer RNA helps match that message to amino acid building blocks; see NHGRI: transfer RNA. Ribosomal RNA forms structural and catalytic parts of the ribosome. Other RNA participates in gene regulatory networks, changing how strongly genes are expressed. Many RNA molecules therefore function without being translated into proteins; for some genes, functional RNA is the product itself.

Most strikingly, some folded RNA acts as an enzyme: these catalytic RNA molecules are ribozymes. The ribosome's bond-forming active site is built from RNA, although the complete ribosome also contains proteins. Structural evidence appears in Nissen et al., The structural basis of ribosome activity in peptide bond synthesis, Science 289, 920–930 (2000). RNA therefore connects a readable record with physical recognition, regulation, and catalysis: the same kind of polymer can be a message, an adaptor, or part of molecular machinery.

Can a gene from Schizosaccharomyces pombe replace a missing function in Saccharomyces cerevisiae? A modern laboratory project tested functional complementation of methionine synthesis. For each tested gene deletion, the investigators compared plasmids carrying the corresponding Saccharomyces cerevisiae gene, a candidate Schizosaccharomyces pombe gene, and an unrelated gene. The first was a positive scientific control; the last was a negative scientific control. The paper describes this design rather than publishing a complete set of individual growth measurements. ^[ref]

The recipient biological strains also lacked the ability to make uracil. The pYES2.1/V5-His plasmids carried URA3, a gene restoring that ability, and used the GAL1 gene promoter to control the inserted gene. Initial plates lacked uracil but supplied methionine: this selected biological colonies containing plasmids without requiring successful methionine synthesis. Subsequent plates lacking methionine tested the missing function, comparing galactose induction with glucose conditions. Thus selecting for a plasmid and testing its functional contribution were separate operations. ^[ref]

The apparatus can be simple and informative. In the associated protocol, replica plating uses a block covered with velveteen to transfer small samples of biological colonies from a master plate onto plates with different nutrient conditions. Orientation marks preserve correspondence between locations; gentle contact limits the transferred material. The specified replicas omit uracil and methionine with either inducing sugar or glucose, and include a fresh glucose plate supplying methionine. The readout is growth at corresponding locations, not a direct measurement of enzyme activity. ^[ref]

The scientific controls answer different objections. Growth on the methionine-supplied replica checks survival and transfer. Rescue by the native gene checks that this biological strain and assay can reveal functional complementation. The unrelated gene tests whether carrying a plasmid alone is sufficient. Comparing induction conditions checks dependence on gene expression, although changing the sugar also changes metabolism: it is not an isolated change of gene expression in every other respect.

If the candidate gene restores growth while those comparisons behave as intended, it supplies a function sufficient for the tested growth requirement in this recipient. Failure to rescue is less decisive: the candidate protein might not be produced, might be altered by its attached tag, or might not work in this cellular context. The investigators therefore also used Western blots to examine protein production. A detected protein still does not establish correct protein folding or catalytic activity. ^[ref]