Non-canonical base pairs are planar, hydrogen-bonded pairs of nucleobases with hydrogen-bonding patterns that differ from those of standard Watson–Crick base pairs found in the classic double-helical structure of DNA. Although non-canonical pairs can occur in both DNA and RNA, they primarily form stable structures in RNA, where they contribute to its structural diversity and functional complexity. In DNA, such base pairs are typically transient and arise during processes like DNA replication.

Each nucleobase presents a unique distribution of hydrogen bond donors and acceptors across three edges: the Watson–Crick edge, the Hoogsteen edge (or C-H edge in pyrimidines), and the sugar edge. Canonical base pairs form through hydrogen bonding along the Watson–Crick edges, while non-canonical pairs often involve the Hoogsteen or sugar edges.

Common types of non-canonical base pairs in RNA include the G:U wobble pair, sheared G:A pair, reverse Hoogsteen A:U pair, and G:A imino pair. Together, these alternative pairings account for roughly one-third of all base pairs in functional RNA structures. The G:U wobble pair, in particular, is abundant in tRNA anticodon loops and facilitates flexible codon recognition. Sheared G:A and reverse Hoogsteen A:U pairs commonly stabilize loops, junctions, and recurrent 3D motifs such as GNRA tetraloops.

Non-canonical base pairs are often located in loops, bulges, and junctions of RNA, where they help stabilize three-dimensional structures and mediate tertiary interactions. They play critical roles in RNA folding, molecular recognition, and catalysis.

Importance

Non-canonical base pairing is indispensable for RNA's structural and functional versatility, enabling the formation of complex three-dimensional architectures essential for biological activity. These alternative pairings, constituting approximately 33% of base pairs in functional RNA structures, stabilize loops, bulges, and junctions that are interspersed between canonical double-helical regions, facilitating the assembly of intricate tertiary folds. They act as molecular "stitches," connecting disparate structural elements while serving as recognition sites for ligands, proteins, and other RNAs. Critically, non-canonical pairs like G:U wobble and sheared G:A underpin recurrent 3D motifs (e.g., kink-turns) and mediate long-range tertiary contacts through higher-order multiplexes, such as base triples observed in over 10% of RNA base pairs. This geometric diversity allows RNA to adopt conformations crucial for catalysis (ribozymes), genetic regulation (riboswitches), and viral replication mechanisms, as seen in HIV Rev peptide-induced structural rearrangements. Without these interactions, RNA would lack the structural plasticity required for its multifunctional roles in cellular processes.

Structural background

An estimated 60% of bases in structured RNA participate in canonical Watson-Crick base pairs. Base pairing occurs when two bases form hydrogen bonds with each other. These hydrogen bonds can be either polar or non-polar interactions. The polar hydrogen bonds are formed by N-H...O/N and/or O-H...O/N interactions. Non-polar hydrogen bonds are formed between C-H...O/N.

Edge interactions

Each base has three potential edges where it can interact with another base. The Purine bases have 3 edges which are able to hydrogen bond. Those are known as the Watson-Crick edge(WC), the Hoogsteen edge(H), and the Sugar edge(S). Pyrimidine bases also have three hydrogen-bonding edges. Like the purine, there is the Watson-Crick edge(WC) and the Sugar edge(S) but the third edge is referred to as the "C-H" edge(H) on the pyrimidine bases. This C-H edge is sometimes also referred to as the Hoogsteen edge for simplicity. The various edges for the purine and pyrimidine bases are shown in Figure 2.

Besides the three edges of interaction, base pairs can also vary in their cis/trans forms. The cis and trans structures depend on the orientation of the ribose sugar as opposed to the hydrogen bond interaction. These various orientations are shown in Figure 3. Therefore, with the cis/trans forms and the 3 hydrogen bond edges, there are 12 basic types of base pairing geometries which can be found in RNA structures. Those 12 types are WC:WC (cis/trans), WC:HC (cis/trans), WC:S (cis/trans), H:S (cis/trans), H:H (cis/trans), and S:S (cis/trans).

Common types The most common non-canonical base pairs are trans A:G Hoogsteen/sugar edge, A:U Hoogsteen/WC, and G:U Wobble pairs.

Hoogsteen base pairs

Hoogsteen base pairs occur between adenine (A) and thymine (T); and guanine (G) and cytosine (C); similarly to Watson-Crick base pairs. However, the purine (A and G) takes on an alternative conformation with respect to the pyrimidine. In the A-U Hoogsteen base pair, the adenine is rotated 180° about the glycosidic bond, resulting in an alternative hydrogen bonding scheme which has one hydrogen bond in common with the Watson-Crick base pair (adenine N6 and thymine N4), while the other hydrogen bond, instead of occurring between adenine N1 and thymine N3 as in the Watson-Crick base pair, occurs between adenine N7 and thymine N3 (the Hoogsteen face). The A-U base pair is shown in Figure 5. In the G-C Hoogsteen base pair, like the A-T Hoogsteen base pair, the purine (guanine) is rotated 180° about the glycosidic bond while the pyrimidine (cytosine) remains in place. One hydrogen bond from the Watson-Crick base pair is maintained (guanine O6 and cytosine N4) and the other occurs between guanine N7 and a protonated cytosine N3 (note that the Hoogsteen G-C base pair has two hydrogen bonds, while the Watson-Crick G-C base pair has three).

Wobble base pairs

Wobble base pairing occur between two nucleotides that are not Watson-Crick base pairs and was proposed by Watson in 1966. They occur between the Watson-Crick faces of bases. The 4 main examples are guanine-uracil (G-U), hypoxanthine-uracil (I-U), hypoxanthine-adenine (I-A), and hypoxanthine-cytosine (I-C). These wobble base pairs are very important in tRNA. Most organisms have less than 45 tRNA molecules even though 61 tRNA molecules would technically be necessary to canonically pair to the codon. Wobble base pairing allows for the 5' anticodon to bond to a non-standard base pair. Examples of wobble base pairs are given in Figure 6.

Classification

Two bases may approach each other in various ways, eventually leading to specific molecular recognition mediated by, often non-canonical, base pairing interactions, in addition to strong stacking interactions. These are essential for the process of RNA single strands folding into three-dimensional structures. Early studies on such unusual base pairs by Jiri Sponer, Pavel Hobza and their group were somewhat disadvantaged due to the unavailability of suitable unambiguous systematic naming schemes. While some of the observed base pair were assigned names following the Saenger nomenclature scheme. others were arbitrarily assigned names by different researchers.  It may be mentioned that some attempts were also made by Michael Levitt and coworkers to classify base-base association in terms of adjacency of bases, through either pairing or stacking interactions. There was clearly a need for a classification scheme for different types of non-canonical base pairs, which could comprehensively and unambiguously handle newer variants coming up due to the rapid increase in the sampling space. Different approaches which have evolved in response to this need are described below.

Edge families

+ Base Pair Geometries (W = Watson–Crick, H = Hoogsteen, S = Sugar edge)Interacting EdgesGlycosidic BondNomenclatureStrand DirectionW/WCiscWWAntiparallelW/WTranstWWParallelW/HCiscWHParallelW/HTranstWHAntiparallelW/SCiscWSAntiparallelW/STranstWSParallelH/HCiscHHAntiparallelH/HTranstHHParallelH/SCiscHSParallelH/STranstHSAntiparallelS/SCiscSSAntiparallelS/STranstSSParallel

The nucleotide bases are nearly planar heterocyclic moieties, with conjugated pi-electron cloud, and with several hydrogen bonding donors and accepters distributed around the edges, usually designated as W, H or S, based on whether the edges can respectively be involved in forming Watson-Crick base pair, Hoogsteen base pair, or, whether the edge is adjacent to the C2'-OH group of the ribose sugar. Eric Westhof and Neocles Leontis used these edge designations to propose a currently widely accepted nomenclature scheme for base pairs. The hydrogen bonding donor and acceptor atoms could thus be classified in terms of their positioning along their three edges, namely the Watson-Crick or W edge, the Hoogsteen or H edge, and the Sugar or S edge. Since base pairs are mediated through hydrogen bonding interactions based on hydrogen bond donor-acceptor complementarity, this, in turn, provides a convenient bottoms-up approach towards classifying base pair geometries in terms of respective interacting edges of the participating bases. It may be noted that, unlike the Hoogsteen edge of purines, the corresponding edges of the pyrimidine bases do not have any polar hydrogen bond acceptor atom such as N7. However, these bases have C—H groups at their C6 and C5 atoms, which can act as weak hydrogen bond donors, as proposed by Gautam Desiraju. The Hoogsteen edge, hence, is also called Hoogsteen/C-H edge in a unified scheme for designating equivalent positions of purines as well as pyrimidines. Thus, the total number of possible edge combinations involved in base pairing are 6, namely Watson-Crick/Watson-Crick (or W:W), Watson-Crick/Hoogsteen (or W:H), Watson-Crick/Sugar (or W:S), Hoogsteen/Hoogsteen (or H:H), Hoogsteen/Sugar (or H:S) and Sugar/Sugar (or S:S).

In the canonical Watson-Crick base pairs, the glycosidic bonds attaching the N9 (of purine) and N1 (of pyrimidine) of the paired bases with their respective sugar moieties, are on the same side of the mean hydrogen bonding axis, and are hence called Cis Watson-Crick base pairs. However, the relative orientations of the two sugars may also be Trans with respect to the mean hydrogen bonding direction giving rise to a distinct Trans Watson-Crick geometric class, consisting of species which were earlier referred to as reverse Watson-Crick base pairs according to Saenger nomenclature. The possibility of both Cis and Trans glycosidic bond orientation for each of the 6 possible edge combinations, gives rise to 12 geometric families of base pairs (see table).

Note on local strand direction