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Base pair

A base pair is a fundamental unit in the structure of nucleic acids, consisting of two complementary nitrogenous bases linked by hydrogen bonds that stabilize the double helix in DNA or contribute to folding in RNA.^[1]^[2] In DNA, the four nucleotide bases—adenine (A), thymine (T), guanine (G), and cytosine (C)—pair specifically: A with T through two hydrogen bonds and G with C through three hydrogen bonds, ensuring uniform spacing and structural integrity of the double helix as elucidated by James D. Watson and Francis H. C. Crick in their 1953 model.^[3]^[4]^[5] This complementary pairing, where purines (A and G) bond with pyrimidines (T and C), positions the bases inward while sugar-phosphate backbones form the outer rails of the helical ladder.^[4] In RNA, which is typically single-stranded, the base composition shifts with uracil (U) substituting for thymine; thus, A pairs with U via two hydrogen bonds, and G pairs with C via three, facilitating intramolecular folding into complex secondary structures such as stem-loops and pseudoknots.^[6]^[7] Base pairing underpins critical biological processes, including DNA replication, where each parental strand templates the synthesis of a complementary daughter strand via semiconservative mechanisms, preserving genetic fidelity across cell divisions.^[5]^[4] In transcription, base pairing between DNA and nascent RNA ensures accurate copying of genetic information, while in RNA molecules like transfer RNA and ribosomal RNA, it enables precise codon-anticodon interactions during protein synthesis.^[7] Non-canonical base pairs, such as G-U wobbles, further diversify RNA structures and functions in regulatory roles.^[8] The human genome, for instance, comprises approximately 3 billion such base pairs distributed across 23 chromosome pairs, underscoring their scale in encoding life's blueprint.^[1]

Fundamentals

Definition and Occurrence

A base pair consists of two complementary nitrogenous bases—one purine and one pyrimidine—held together by hydrogen bonds within the structure of double-stranded nucleic acids. The purines are adenine (A) and guanine (G), while the pyrimidines are cytosine (C), thymine (T) in DNA, or uracil (U) in RNA. These bases form the core of nucleotides, where each base is covalently linked to a sugar molecule (deoxyribose in DNA or ribose in RNA) to create a nucleoside, which is then incorporated into the polynucleotide chain.^[4] The concept of base pairing was first proposed by James D. Watson and Francis H. C. Crick in their 1953 description of the DNA double helix, where they identified specific pairings of A with T and G with C as essential to the molecule's structure and function. This model provided a mechanism for genetic replication, as the sequence of bases on one strand determines the complementary sequence on the other. The principle was soon extended to RNA, where U substitutes for T in pairing with A, enabling the formation of double-stranded regions in RNA molecules.^[3]^[9] Base pairs occur primarily in the antiparallel double helix of DNA, which adopts the right-handed B-form conformation characterized by a smooth, uniform twist with approximately 10.5 base pairs per helical turn. In RNA, base pairing is found in double-stranded segments of secondary structures, such as the stems of hairpins and loops, forming A-form helices that are shorter and wider than the B-form due to the 2'-hydroxyl group on ribose. These pairings are crucial for storing genetic information in DNA, facilitating its accurate replication during cell division, and supporting RNA functions in transcription and translation for protein synthesis.^[4]^[10]

Canonical Base Pairs

Canonical base pairs refer to the standard Watson-Crick pairings that form the foundation of double-stranded nucleic acids, consisting of adenine (A) with thymine (T) in DNA or uracil (U) in RNA, and guanine (G) with cytosine (C). These pairs occur between a purine base on one strand and a pyrimidine base on the complementary strand, maintaining consistent width in the double helix. The adenine-thymine (A-T) or adenine-uracil (A-U) pair forms through two hydrogen bonds: the N1 of adenine bonds to N3 of thymine or uracil, and the O4 (or O2 in uracil) of thymine/uracil bonds to the amino group at C6 of adenine. In contrast, the guanine-cytosine (G-C) pair involves three hydrogen bonds: O6 of guanine to amino at C4 of cytosine, N1 of guanine to N3 of cytosine, and amino at C2 of guanine to O2 of cytosine. This specific hydrogen bonding pattern, along with the complementary shapes of the bases, ensures precise alignment within the helical structure.^[11]^[12] The geometry of these pairs positions the bases perpendicular to the helix axis, fitting snugly into the major and minor grooves while allowing the sugar-phosphate backbones to form the outer scaffold. The G-C pair's three hydrogen bonds provide greater stability than the A-T/U pair's two, influencing the overall melting temperature of nucleic acid duplexes, though this difference arises directly from the bonding count. Chargaff's rules underpin the equivalence observed in base compositions of double-stranded DNA, stating that the proportion of adenine equals thymine (A = T) and guanine equals cytosine (G = C), a direct consequence of the complementary pairing across strands. These rules were established through quantitative analyses of DNA from various organisms, revealing species-specific but internally balanced base ratios. In double-stranded RNA, similar equivalence holds with A = U and G = C.^[13] A key distinction between DNA and RNA canonical pairing lies in the use of thymine versus uracil. DNA employs thymine to pair with adenine, offering enhanced resistance to spontaneous cytosine deamination (which produces uracil).^[14] It also provides better protection against UV-induced photodimers, as uracil is more prone to such damage.^[15] Thymine also facilitates 5-methylcytosine formation for epigenetic marking without confusing repair systems. RNA, being shorter-lived and single-stranded in many contexts, uses uracil, which is energetically cheaper to synthesize as it derives directly from orotate without methylation. Despite this substitution, A-U pairing mirrors A-T in hydrogen bonding and specificity. The resulting duplexes exhibit subtle structural variations: DNA favors the right-handed B-form helix with ~10.5 base pairs per turn and a wide major groove, while RNA duplexes adopt the A-form with ~11 base pairs per turn, a narrower major groove, and greater base tilting due to the 2'-hydroxyl group on ribose, yet both preserve the canonical pairing geometry.^[14]^[16] The specificity of canonical base pairs is crucial for fidelity in genetic processes, as the unique hydrogen bonding sites and steric complementarity prevent mismatched pairings, such as A-C or G-T, which would distort the helix and lead to replication or transcription errors. This selective recognition enables accurate information transfer, with purine-pyrimidine matching ensuring uniform helix dimensions and groove accessibility for proteins.^[11]

Notation

In scientific literature, base pairs are conventionally denoted using single-letter symbols for the nucleobases: adenine (A) pairs with thymine (T) in DNA or uracil (U) in RNA, while guanine (G) pairs with cytosine (C) in both, as established by the Watson-Crick model.^[1] These pairings are often represented with hyphens or lines to indicate hydrogen bonding, such as A-T or G-C for DNA and A-U or G-C for RNA.^[17] For nucleotide sequences, double-stranded DNA or RNA is typically written in a 5' to 3' direction for the forward strand, with the complementary strand shown in the antiparallel 3' to 5' orientation, connected by lines or spaces to highlight pairings; for example, the sequence 5'-ATGC-3' pairs with 3'-TACG-5'.^[18] This convention uses the International Union of Pure and Applied Chemistry (IUPAC) single-letter codes, where A denotes adenine, C cytosine, G guanine, T thymine (or U for uracil in RNA), ensuring standardized representation across diagrams and sequences.^[19] In structural diagrams, base pairs are illustrated following the Watson-Crick model, depicting antiparallel strands as parallel lines or ribbons with horizontal rods or bonds connecting the paired bases, emphasizing their orientation and complementarity without detailing bond specifics.^[20] To handle ambiguity in mixed DNA/RNA contexts or uncertain bases, IUPAC ambiguity codes are employed, such as Y for pyrimidines (C, T, or U), R for purines (A or G), and N for any base (A, C, G, T/U).^[21] The notation evolved historically from Erwin Chargaff's 1940s observations of base composition equalities (A ≈ T, G ≈ C) in DNA, which informed Watson and Crick's 1953 proposal of specific pairings, leading to modern bioinformatics formats like FASTA, where paired sequences are represented by separate entries for each strand with implied complementarity.^[22]^[23]

Chemical Properties

Hydrogen Bonding

Hydrogen bonding serves as the primary chemical interaction stabilizing canonical base pairs in nucleic acids, involving electrostatic attractions between a hydrogen atom covalently bound to an electronegative atom (typically nitrogen or oxygen) acting as a donor and another electronegative atom serving as an acceptor.^[3] This donor-acceptor mechanism ensures specific pairing between complementary bases, with the hydrogen bonds forming between precise atomic sites on the purine and pyrimidine rings. For instance, in the adenine-thymine (A-T) or adenine-uracil (A-U) pair, bonds occur between the N1 of adenine (acceptor) and the N3-H of thymine/uracil (donor), as well as between the N6-H of adenine (donor) and the O4 of thymine/uracil (acceptor).^[11] Similarly, in the guanine-cytosine (G-C) pair, three bonds form: O6 of guanine (acceptor) to N4-H of cytosine (donor), N1-H of guanine (donor) to N3 of cytosine (acceptor), and N2-H of guanine (donor) to O2 of cytosine (acceptor).^[11] The number of hydrogen bonds differs between pairs, contributing to their relative strengths: two bonds in A-T/U and three in G-C, which promotes the observed base composition biases in DNA sequences.^[3] These interactions occur exclusively via the Watson-Crick edges of the bases, where the donor and acceptor sites align in a complementary fashion to maximize bond formation without steric clashes.^[24] Geometrically, the hydrogen bonds enforce a planar configuration of the base pairs, with the glycosidic bonds adopting an anti-parallel orientation relative to the sugar-phosphate backbones, ensuring uniform helical parameters in the double helix.^[24] This planarity arises from the sp² hybridization of the ring atoms involved, allowing the bases to lie flat and stack efficiently while the bonds hold them in register.^[3] From a quantum mechanical perspective, each hydrogen bond in these pairs has an energy of approximately 5-30 kJ/mol, reflecting partial covalent character and directionality that enhances specificity.^[25] The complementary hydrogen-bonding patterns, dictated by the predominant keto and amino tautomeric forms of the bases, ensure selective pairing; for example, the keto form of thymine provides the necessary O4 acceptor, while rare enol tautomers could disrupt this fidelity but are minimized in vivo.^[3] These patterns create a lock-and-key-like recognition, where mismatches result in suboptimal bonding geometries and energies.^[26] In aqueous environments, solvent molecules like water compete for hydrogen-bonding sites on the bases, weakening individual inter-base bonds by stabilizing the lone pairs and hydrogens involved, often leading to slight lengthening of bond distances. However, this competition is counterbalanced by the overall stabilization of the double helix through desolvation effects and the hydrophobic burial of bases, maintaining the integrity of the paired structure.

Stability Factors

The stability of base pairs in nucleic acid duplexes is influenced by several factors beyond hydrogen bonding, with base stacking emerging as a dominant contributor through hydrophobic and π-π interactions between adjacent base pairs along the helix axis. These stacking interactions, which involve the overlap of aromatic rings in the bases, provide the majority of the duplex's thermal stability, accounting for approximately 50-70% of the overall free energy stabilization in double-stranded DNA.^[27] Sequence dependence plays a key role here, as purine-pyrimidine stacks like those involving guanine-cytosine (GC) exhibit stronger interactions due to better orbital overlap and higher electron density compared to adenine-thymine (AT) stacks, leading to enhanced stability in GC-rich regions.^[27] Electrostatic interactions also significantly affect base pair durability, primarily through the repulsion between negatively charged phosphate groups in the sugar-phosphate backbone. This repulsion, which can destabilize the duplex by up to 30% of the energy required for structural deformations like bending, is counterbalanced by the screening effects of cations such as Na⁺ and Mg²⁺, which condense around the phosphates to neutralize charges and reduce the overall electrostatic penalty.^[28] Additionally, a dehydration penalty arises during duplex formation, as the hydrophobic bases must exclude water molecules from their interior, contributing an entropic cost that is partially offset by the release of structured water from the grooves.^[29] The conformational context of the helix further modulates stability, with distinct parameters for B-DNA and A-form RNA influencing base pair accessibility and interactions. In B-DNA, the right-handed helix features approximately 10.5 base pairs per turn and a rise of 0.34 nm per base pair, resulting in a wider major groove (about 1.2 nm) that exposes edges of the bases for interactions, while the minor groove is narrower (0.6 nm).^[30] In contrast, A-form RNA adopts a more compact structure with 11 base pairs per turn and a rise of 0.26 nm per base pair, producing a deep, narrow major groove (~0.3 nm wide, 1.3 nm deep) and a shallow, wide minor groove (~1.1 nm wide, 0.3 nm deep), which limits solvent access and enhances stacking efficiency but can hinder protein binding.^[30] These geometric differences affect the overall rigidity and environmental sensitivity of the duplex. Ionic strength significantly influences stability; higher salt concentrations screen phosphate repulsions, raising the melting temperature (Tm) according to relations like ΔTm ≈ 16.6 log₁₀([Na⁺]/0.1 M) °C.^[31] Thermodynamically, base pair stability is quantified through parameters that capture the energetic contributions of these interactions. The enthalpy change (ΔH) primarily arises from hydrogen bonds and base stacking, typically ranging from -7 to -10 kcal/mol per base pair, while the entropy change (ΔS) reflects the ordering of strands and loss of solvent freedom, often negative at around -20 to -25 cal/mol·K per base pair. The Gibbs free energy of duplex formation is given by

\Delta G = \Delta H - T \Delta S

where T is the temperature in Kelvin; this relation underpins predictions of the melting temperature (Tm), the point at which half the duplex dissociates, with higher stability correlating to elevated Tm values. Sequence composition exerts a profound influence on these thermodynamic properties, particularly through GC content, which elevates Tm by 0.4–0.5°C per 1% increase due to the three hydrogen bonds in GC pairs and their superior stacking strength compared to the two-bond AT pairs. This effect is evident in polymers where poly(dG·dC) exhibits a Tm approximately 30–40°C higher than poly(dA·dT) under similar ionic conditions, underscoring the role of base identity in modulating duplex resilience.

Examples

One illustrative measure of base pair stability is the melting temperature (Tm), the temperature at which half of the double-stranded DNA dissociates into single strands, which can be approximated for short oligonucleotides in ~1 M NaCl using the empirical equation T_m = 69.3 + 0.41 \times (\%GC), where %GC is the percentage of guanine-cytosine base pairs.^[32] This formula highlights the stabilizing effect of G-C pairs, which contribute more to Tm than A-T pairs due to their additional hydrogen bond. For example, under low salt conditions (e.g., 10 mM Na⁺), poly(dA-dT) sequences exhibit a relatively low Tm of approximately 39°C, reflecting the weaker stability from two hydrogen bonds per pair, whereas poly(dG-dC) sequences display a high Tm around 94°C, underscoring the robustness from three hydrogen bonds.^[33] In higher salt (e.g., 0.2 M NaCl), these values increase, with poly(dA-dT) around 65°C and poly(dG-dC) over 100°C. The nearest-neighbor model provides a more detailed prediction of duplex stability by considering the additive effects of adjacent base pair stacks, with parameters derived from experimental thermodynamic data. In this model, the free energy of stacking interactions varies such that AA/TT stacks are weaker (less stable) compared to GG/CC stacks, which are among the strongest, allowing for accurate Tm predictions within about 2°C for diverse sequences. These parameters, compiled in SantaLucia tables, account for sequence-specific contributions beyond simple %GC content. Environmental factors further modulate base pair stability, as seen in the influence of salt concentration and pH on Tm. Increasing salt concentration raises Tm by shielding the negative charges on phosphate backbones, reducing electrostatic repulsion between strands and thereby enhancing duplex stability. At low pH, protonation of cytosine (with a pKa around 4.5) disrupts G-C pairing by introducing positive charges that alter hydrogen bonding patterns and increase repulsion.^[34] In pathological contexts, the triple hydrogen bonds of G-C pairs contribute to structural transitions, such as the formation of left-handed Z-DNA in G-C-rich sequences under high salt conditions, where the zigzag backbone conformation is stabilized by the dense bonding network.^[35] For RNA, base pair stability is exemplified in tRNA stem-loops, where short stretches of canonical Watson-Crick pairs maintain structural integrity primarily through base stacking interactions, enabling functional folding even with limited hydrogen bonding.^[36] Stacking and hydrogen bonding, as outlined in prior sections, underpin these examples by providing the energetic basis for observed stability variations.

Variations

Non-Canonical Base Pairing

Non-canonical base pairing involves hydrogen-bonded interactions between nucleobases that deviate from the standard Watson-Crick geometry, often utilizing alternative faces such as the Hoogsteen edge of purines or the sugar edge, enabling structural flexibility in nucleic acids.^[37] Common types include Hoogsteen pairs, where a purine uses its Hoogsteen face to pair with a pyrimidine's Watson-Crick face, forming two or three hydrogen bonds; reverse Hoogsteen pairs, which invert this orientation; sheared pairs, characterized by parallel strand geometry and sugar-edge interactions, such as the sheared G:A pair; and wobble pairs, featuring a shifted alignment with typically two hydrogen bonds, exemplified by the G:T in DNA or G:U in RNA.^[38] These pairings contrast with canonical Watson-Crick pairs by promoting adaptability in folding and function, though they are less prevalent overall.^[39] In DNA, non-canonical base pairs frequently arise as mismatches during replication or repair processes, such as the A:C mismatch, which adopts conformations like a protonated C paired with A via two hydrogen bonds in repair intermediates, influencing recognition by mismatch repair enzymes.^[40] The G:T wobble pair, with its two hydrogen bonds and displaced geometry, also occurs in such contexts, contributing to transient instabilities that trigger correction mechanisms.^[41] Additionally, Hoogsteen and reverse Hoogsteen pairs appear in Holliday junctions during homologous recombination, where they facilitate branch migration and structural isomerization, as seen in four-way DNA junctions with non-canonical G:C Hoogsteen pairings that induce kinking.^[42] In RNA, non-canonical base pairs are more abundant and integral to tertiary structure formation, particularly in loops and motifs. The G:U wobble pair, stabilized by two hydrogen bonds between guanine's Watson-Crick face and uracil's Hoogsteen face, is ubiquitous and plays key roles in stabilizing tRNA anticodon loops and ribosomal RNA structures.^[43] Sheared G:A pairs, involving sugar-edge contacts, and reverse Hoogsteen A:U pairs commonly occur in these regions, enabling compact folds in functional RNAs like ribozymes.^[38] While many non-canonical pairs exhibit lower stability than canonical ones due to fewer hydrogen bonds (typically 1-2 versus 2-3 in some cases), resulting in higher free energies and greater susceptibility to disruption, wobble pairs like G:U often have stability comparable to Watson-Crick pairs (around 80-100% depending on context) owing to similar hydrogen bonding and stacking.^[44] Surrounding stacking interactions and ionic environments can compensate to maintain viability in context-specific roles like triplex or quadruplex formation. For instance, in duplex contexts, G:U wobble contributions are around -7 to -10 kcal/mol, similar to A:U pairs.^[45] Detection of non-canonical base pairs relies on high-resolution techniques such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, which reveal their geometries through atomic coordinates and chemical shift patterns. Cryo-electron microscopy has recently enabled visualization of such pairs in large complexes (as of 2025).^[46] In analyzed RNA structures from crystal databases, non-canonical pairs account for approximately 30-40% of total interactions, with wobble and sheared types being most frequent.^[47] NMR relaxation dispersion further identifies transient Hoogsteen forms in DNA duplexes at rates up to milliseconds.^[48]

Wobble Pairs and RNA-Specific Interactions

The wobble hypothesis, proposed by Francis Crick in 1966, posits that the third position of the codon-anticodon interaction during translation allows for non-standard base pairing, thereby accommodating degeneracy in the genetic code without requiring a unique tRNA for each codon.^[49] Specifically, uracil (U) in the anticodon's first position can pair with adenine (A) or guanine (G) in the codon's third position, while inosine (I), a modified base common in tRNA anticodons, can pair with uracil (U), cytosine (C), or adenine (A).^[49] This flexibility arises from the structural geometry of the wobble pairs, which permit hydrogen bonding despite deviations from strict Watson-Crick rules, enabling a single tRNA to recognize multiple synonymous codons. In RNA, wobble and other non-canonical interactions contribute to the formation of complex motifs essential for structural stability and function. Pseudoknots, for instance, feature interlocking helical stems where non-canonical pairs, including wobbles, bridge loops and adjacent regions to create tertiary architectures critical for processes like ribosomal frameshifting. Base triples in ribosomal RNA often involve a wobble pair in a helix interacting with a third base from a distant strand, facilitating long-range contacts that assemble the ribosome's functional core. G-quadruplexes in RNA, while primarily stabilized by Hoogsteen hydrogen bonds and stacking interactions rather than pairwise canonical or wobble pairing, incorporate non-canonical elements that enhance folding in guanine-rich sequences, influencing RNA localization and regulation. These interactions underpin key biological roles in RNA functionality. The wobble hypothesis directly enables the decoding of 61 sense codons using fewer than 61 tRNAs, optimizing translational efficiency across organisms.^[49] In ribozymes, wobble pairs provide structural plasticity; for example, in the hammerhead ribozyme, GU wobble pairs within the core maintain catalytic competence by allowing conformational adjustments during self-cleavage, while allosteric variants use ligand-induced stabilization of wobble pairs to enhance activity. Similarly, in the hepatitis delta virus (HDV) ribozyme, multiple GU wobbles contribute to the active site's stability, with mutations disrupting them impairing cleavage rates. Wobble pairs exhibit thermodynamic stability comparable to Watson-Crick pairs, often around 80-100% of their strength depending on sequence context, due to similar hydrogen bonding patterns and stacking energies. In flexible RNA structures, they are entropy-favored, as the looser geometry permits greater conformational freedom, reducing energetic penalties in dynamic environments like tRNA-mRNA interactions. Recent studies have expanded understanding of wobble and related non-canonical interactions in RNA regulation. In microRNAs (miRNAs), pairing beyond the canonical seed region (positions 2-8) via wobble or mismatch-tolerant modes at positions 9-13 enhances target specificity and efficacy, as revealed by abasic modifications and structural mapping in 2025 experiments.^[50] Dynamics of U-U mismatches, a type of non-canonical pair, in A-form RNA helices show sequence-dependent flexibility, where local strain from the mismatch promotes base flipping and helix breathing; such effects influence viral RNA stability, as observed in SARS-CoV-2 structures via molecular dynamics simulations in 2024^[51] and general RNA contexts in 2025.^[52]

Synthetic and Unnatural Pairs

Development of Unnatural Base Pairs

The development of unnatural base pairs (UBPs) aimed to expand the genetic alphabet beyond the canonical A-T and G-C pairs, enabling new biological functions such as encoding additional amino acids or creating novel diagnostic tools. Early efforts in the 1960s focused on hydrogen-bonding mimics that could form stable, orthogonal pairs without interfering with natural bases. In 1962, Alexander Rich proposed the isoguanine (isoG)-isocytosine (isoC) pair, which features three hydrogen bonds similar to G-C, suggesting it could serve as a third base pair in an expanded genetic system. By the 1990s, synthetic chemistry advanced these concepts, with Steven Benner's group synthesizing isoG and isoC nucleosides and demonstrating their incorporation into oligonucleotides, enzymatic replication, and even in vitro translation using a dedicated codon, though challenges like chemical instability (e.g., isoG deamination to xanthine) and tautomerism reduced selectivity to around 93% per PCR cycle. Concurrently, Eric Kool's group introduced hydrophobic pairs, such as the nonpolar difluorotoluene (F)-thymine mimic in 1997, emphasizing shape complementarity and pi-stacking over hydrogen bonding to achieve pairing stability in DNA duplexes without relying on traditional H-bonds. Key UBP systems emerged in the 2000s, prioritizing orthogonality to natural bases for reliable replication by polymerases. Benner's group developed the 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (P) and 6-amino-5-nitropyridin-2(1H)-one (Z) pair around 2003-2006, using a non-standard hydrogen-bonding pattern to achieve up to 99.9% fidelity in PCR amplification after optimizations. In 2006, Ichiro Hirao's group reported the 7-(2-thienyl)imidazo[4,5-b]pyridine-2(3H)-one (Ds)-pyrrole-2-carbaldehyde (Pa) pair, a hydrophobic system that relies on minor-groove interactions and achieves >99% selectivity per replication cycle when using modified triphosphates. Floyd Romesberg's group introduced the 5-(6-aminopyridin-3-yl)-2'-deoxyuridine-5'-triphosphate (d5SICS) and 2-amino-8-(2-thienyl)purine (NaM) pair in the early 2010s, designed for high orthogonality and efficient polymerase-mediated replication with fidelities up to 99.8%.^[53] Design principles for these UBPs emphasize geometric fit within the DNA helix and avoidance of natural base interference, often favoring hydrophobic and pi-stacking forces over hydrogen bonding to minimize mispairing, while ensuring recognition by cellular enzymes through subtle modifications like halogen substitutions or fused rings.^[54] A major milestone came in 2014, when Romesberg's team engineered an E. coli strain to stably replicate and transcribe DNA containing the d5SICS-NaM pair, creating the first semi-synthetic organism with a six-letter genetic alphabet. Building on this, in 2019, a collaboration between Romesberg and Benner's groups developed "hachimoji" DNA, an eight-letter system incorporating two orthogonal UBPs (P-Z and S-B) alongside natural bases, which forms stable duplexes and supports PCR amplification in vitro, paving the way for more complex synthetic genetics.^[55] Despite progress, challenges persist in achieving consistent enzymatic fidelity across diverse polymerases and in vivo contexts, as UBPs can compete with natural substrates, leading to retention rates as low as 80% in early cellular uptake experiments. Additionally, imbalances in unnatural nucleotide triphosphate pools can cause cellular toxicity by perturbing natural DNA synthesis and inducing mutations.^[56]

Recent Advances and Applications

In 2025, researchers developed an unnatural base pair system utilizing the MfC:D pair for bisulfite-free detection of the epigenetic modification 5-formylcytosine (5fC) in DNA sequencing, with potential extension to 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) via chemical conversion.^[57] This pair achieves enhanced duplex stability through three hydrogen bonds, allowing selective incorporation opposite modified cytosines without the DNA degradation associated with traditional bisulfite methods.^[57] The approach facilitates base-resolution analysis of these markers, advancing epigenetic profiling in complex genomes.^[57] Recent studies in 2024 have explored metal-mediated unnatural base pairs derived from imidazole nucleobases, which coordinate with ions like Cu²⁺ or Ag⁺ to provide tunable stability in DNA duplexes. These pairs enable dynamic control of hybridization strength by varying metal concentration or type, with Ag⁺-mediated imidazole pairs demonstrating reversible switching in DNAzyme activity for sensor applications. Such systems offer precise modulation of nucleic acid structures, with thermal stabilities adjustable over a range of 10–20°C depending on the metal ligand. Unnatural base pairs have expanded applications in synthetic biology, notably by enabling an eight-letter genetic alphabet that supports 512 possible codons for incorporating non-standard amino acids into proteins.^[55] This expansion, building on systems like d5SICS-NaM, allows site-specific insertion of diverse functionalities during translation, enhancing protein engineering for therapeutic designs. In aptamer engineering, unnatural base pair mutants have been integrated to boost binding affinity and specificity.^[58] Further applications include xeno-nucleic acids (XNAs), synthetic polymers with unnatural backbones like threose or arabinose that pair with unnatural bases for orthogonal replication.^[59] These ubp-XNAs enable high-fidelity nanopore sequencing and evolution of novel enzymes resistant to natural nucleases, expanding the toolkit for in vitro selection.^[59] In biosensor development, unnatural base pair variants have optimized ligand detection platforms, reducing response times in fluorescence-based assays for small molecules by enhancing structural dynamics.^[58] Emerging research points to unnatural base pairs' potential in in vivo therapeutics, where stable incorporation supports targeted gene modulation. Additionally, efforts to develop CRISPR-compatible unnatural base pairs aim to enable precise, off-target-free editing by introducing orthogonal pairing in guide RNAs, with preliminary studies showing improved specificity in non-Watson-Crick contexts.^[60]

Biological Roles

Mutations and Mismatches

Base pair mismatches occur primarily during DNA replication or transcription when incorrect nucleotides are incorporated opposite template bases, leading to genetic errors that introduce variation. These mismatches can arise from spontaneous chemical changes in nucleotides or external factors. One key cause is tautomerization, where bases shift between keto and enol (or amino and imino) forms, altering hydrogen bonding patterns; for instance, the enol form of thymine can pair with guanine instead of adenine, resulting in a T-G mispair.^[61] Depurination, the loss of a purine base (adenine or guanine) from the DNA backbone, or apyrimidination, the analogous loss of a pyrimidine, creates abasic sites that increase the likelihood of incorrect base insertion during replication, as the polymerase may insert any nucleotide opposite the gap.^[62] Environmental mutagens, such as ultraviolet radiation or chemical agents like alkylating compounds, further promote mismatches by damaging bases; UV light, for example, induces cyclobutane pyrimidine dimers that distort pairing fidelity upon replication bypass.^[63] Mismatches are classified into two main types based on the chemical nature of the substitution: transitions and transversions. Transitions involve the replacement of one purine by another (adenine to guanine or vice versa) or one pyrimidine by another (cytosine to thymine or vice versa), such as an A-T pair mutating to G-C through an A-to-G change.^[64] Transversions, in contrast, swap a purine for a pyrimidine or vice versa, like an A-T pair becoming C-G via an A-to-C substitution, which often requires more significant structural adjustments in the helix.^[65] These errors occur at a frequency of approximately 10^{-5} mismatches per base pair during eukaryotic DNA replication without proofreading, though proofreading reduces this to approximately 10^{-7}, and MMR further lowers the overall error rate to around 10^{-10} errors per base pair per replication cycle.^[66] The consequences of uncorrected base pair mismatches manifest as point mutations, where a single nucleotide substitution alters the genetic code, potentially leading to amino acid changes (missense mutations), premature stop codons (nonsense mutations), or silent changes.^[67] In cases of polymerase slippage on repetitive sequences, mismatches can also cause small insertions or deletions, resulting in frameshift mutations that disrupt reading frames downstream.^[63] While these mutations drive evolutionary adaptation by generating genetic diversity, they pose risks such as oncogenic transformations in somatic cells, contributing to cancer development when proto-oncogenes or tumor suppressors are affected.^[62] Detection of mismatches relies on the structural distortions they induce in the DNA double helix; an incorrect base pair creates a local "bubble" or bulge that deviates from the standard B-form geometry, making it recognizable by cellular proteins that scan for such anomalies.^[68] Some non-canonical base pairs, like G-U in RNA, can similarly function as transient mismatches during replication or transcription.^[69]

Repair Mechanisms

Cellular repair mechanisms are essential for recognizing and correcting distortions in base pairing caused by replication errors or environmental damage, thereby preserving genomic integrity. These pathways primarily target mismatches, damaged bases, or bulky lesions that disrupt normal Watson-Crick pairing, employing specialized enzymes to excise erroneous segments and resynthesize accurate sequences. In DNA, the main systems include mismatch repair (MMR), base excision repair (BER), and nucleotide excision repair (NER), which collectively reduce replication errors from an initial rate of about 10^{-5} to as low as 10^{-10} per nucleotide.^[70] In RNA, repair is less prevalent but includes editing mechanisms that modify base pairing without excision. Mismatch repair (MMR) operates post-replication to correct base-base mismatches and small insertion/deletion loops that evade proofreading by DNA polymerases. In prokaryotes like Escherichia coli, the process begins with MutS protein recognizing the distortion caused by a mismatched base pair, forming an ATP-bound sliding clamp that diffuses along the DNA to recruit MutL.^[71] MutL then coordinates excision by interacting with MutH endonuclease, which nicks the unmethylated daughter strand at a nearby hemimethylated GATC site, enabling strand-specific repair.^[71] Exonucleases such as ExoI (5'→3') or RecJ (3'→5'), aided by UvrD helicase, remove the segment containing the mismatch, after which DNA polymerase III resynthesizes the gap using the parental strand as a template, and ligase seals the nick.^[71] Strand discrimination in prokaryotes relies on transient hemimethylation by Dam methylase, where the newly synthesized strand remains unmethylated for several minutes, directing repair exclusively to it.^[72] In eukaryotes, homologs such as MSH2/MSH6 (MutSα) and MLH1/PMS2 (MutLα) perform analogous roles, using nicks or PCNA at replication forks for strand bias. Defects in human MMR genes, particularly germline mutations in MLH1 (∼50% of cases) or MSH2 (∼40%), lead to microsatellite instability and hereditary nonpolyposis colorectal cancer, known as Lynch syndrome.^[73]^[73] Base excision repair (BER) addresses single-base damage that alters pairing, such as spontaneous deamination of cytosine to uracil, creating a U·G mismatch. The pathway initiates with a DNA glycosylase, like uracil-DNA glycosylase (UNG), which specifically recognizes and excises the aberrant base by flipping it out of the helix and cleaving the N-glycosidic bond, generating an apyrimidinic (AP) site.^[74] AP endonuclease 1 (APE1) then incises the phosphodiester backbone at the AP site, creating a single-nucleotide gap. DNA polymerase β fills this gap by inserting the correct base (cytosine opposite guanine), and DNA ligase III, often with XRCC1, seals the repair.^[74] This short-patch BER predominates for uracil repair, preventing C·G to T·A transition mutations, and occurs frequently—up to 10,000 times per day in human cells—to counter oxidative and hydrolytic damage.^[74] Nucleotide excision repair (NER) targets bulky, helix-distorting lesions that severely impair base pairing, such as UV-induced cyclobutane pyrimidine dimers (CPDs) or (6-4) photoproducts. Recognition begins with the XPC-RAD23B-CETN2 complex binding to unpaired bases adjacent to the lesion, often aided by UV-damaged DNA-binding protein (UV-DDB) for chromatin remodeling and enhanced detection of CPDs.^[75] TFIIH, containing the XPD helicase, verifies the damage by attempting to unwind the DNA; blockage at the lesion recruits XPA and RPA for stabilization, leading to dual incisions (∼24 nucleotides 5' and ∼5-6 nucleotides 3' to the lesion) by XPG and ERCC1-XPF endonucleases.^[75] The excised oligonucleotide is removed, and the gap is filled by polymerases δ/ε with PCNA, followed by ligation via XRCC1-LIG3 or LIG1. NER operates in two subpathways—global genome NER for non-transcribed regions and transcription-coupled NER for active genes—ensuring efficient removal of UV dimers that would otherwise block replication and transcription.^[75] These DNA repair mechanisms collectively enhance replication fidelity, reducing the intrinsic polymerase error rate of ∼10^{-5} by 100- to 1,000-fold through proofreading and an additional 100- to 1,000-fold via MMR and other pathways, achieving an overall mutation rate of ∼10^{-10} per base pair.^[70] In RNA, repair is rarer and typically involves site-specific editing rather than excision; adenosine deaminases acting on RNA (ADARs), particularly ADAR1 and ADAR2, convert adenosine to inosine in double-stranded regions, which is read as guanosine during translation and base pairs with cytosine like G·C. This A-to-I editing alters codon meaning (e.g., glutamine to arginine) or RNA structure, contributing to transcriptome diversity but not directly correcting mismatches.^[76]

Base Analogs and Intercalators

Base analogs are synthetic nucleoside or nucleotide mimics that can be incorporated into DNA or RNA during replication or transcription, often leading to errors in base pairing. For instance, 5-bromouracil (BrU) serves as an antimetabolite that substitutes for thymine in DNA, typically pairing with adenine like thymine, but under certain conditions, such as enol tautomerization, it pairs with guanine, inducing A-T to G-C transition mutations.^[77]^[78] Another example is azidothymidine (AZT), a thymidine analog that lacks a 3'-hydroxyl group; after phosphorylation to AZT-triphosphate, it is incorporated into nascent DNA by HIV reverse transcriptase, acting as a chain terminator that halts viral DNA synthesis.^[79] Similarly, acyclovir, a guanosine analog, is selectively phosphorylated by viral thymidine kinase and incorporated into herpesvirus DNA, where it terminates chain elongation by inhibiting viral DNA polymerase.^[80] DNA intercalators are planar aromatic molecules that insert between adjacent base pairs of the double helix, distorting its structure and interfering with enzymatic processes. Ethidium bromide, a phenanthridinium derivative, intercalates via π-stacking interactions with bases, unwinding the helix by approximately 26 degrees per bound molecule and increasing the contour length of DNA.^[81] Daunomycin (daunorubicin), an anthracycline antibiotic, similarly inserts between base pairs through its planar aglycone ring, which stabilizes the complex and inhibits topoisomerase II by trapping the enzyme-DNA cleavage complex.^[82] Doxorubicin, a close structural analog of daunomycin, binds DNA with high affinity (Kd ~ 0.1-1 μM), unwinding the helix and promoting DNA strand breaks via topoisomerase II poisoning.^[83] The mechanisms of these agents exploit vulnerabilities in nucleic acid synthesis. Base analogs like BrU and AZT induce point mutations or replication arrest by promoting mispairing or lacking extension sites, respectively, with BrU specifically favoring transition mutations through altered hydrogen bonding.^[78] Intercalators such as ethidium bromide and daunomycin elevate mutation rates by stabilizing non-Watson-Crick base pairs or impeding helicase and polymerase progression, often causing frameshift mutations due to slippage during replication of repetitive sequences.^[84] These distortions can also block transcription and replication forks, indirectly increasing mutagenesis by prolonging exposure to error-prone repair pathways. In applications, base analogs have revolutionized antiviral therapy; AZT was the first approved treatment for HIV, reducing viral load by targeting reverse transcriptase, while acyclovir treats herpes infections with minimal host toxicity due to poor mammalian kinase activation.^[79]^[80] Intercalators like doxorubicin serve as cornerstone anticancer agents, used in regimens for leukemia, lymphoma, and solid tumors, where DNA intercalation disrupts rapidly dividing cancer cell proliferation.^[83] Both classes are employed in mutagenesis studies: BrU and ethidium bromide help map replication fidelity in model organisms by inducing targeted genetic changes.^[78]^[81] Toxicity arises from their genotoxic effects, with intercalators like doxorubicin promoting frameshift mutations and chromosomal aberrations in non-target cells, particularly those undergoing division.^[84] Base analogs such as AZT exhibit mitochondrial toxicity in long-term use, leading to myopathy via inhibited mitochondrial DNA synthesis.^[85] Both agent types induce apoptosis in sensitive cells; for example, doxorubicin triggers caspase activation and cell death through DNA damage signaling pathways, contributing to its therapeutic efficacy but also dose-limiting cardiotoxicity.^[86]

Measurements

As a Structural Unit

In the B-form of DNA, which represents the predominant physiological conformation, each base pair contributes an axial rise of 0.34 nm along the helical axis, with approximately 10 base pairs completing one full turn of the right-handed helix. This uniform spacing results in a helical pitch of 3.4 nm and defines the structural scaffold for genetic information storage. The cross-sectional area of the double helix, with a diameter of roughly 2 nm, yields an approximate volume of 1.1 nm³ per base pair, accounting for the cylindrical geometry of the molecule.^[30]^[87] Helical parameters further characterize the base pair as a structural unit, including the twist angle of 36° per base pair in B-DNA, which orients successive pairs relative to the helix axis. Local variations are described by roll (rotation about the long axis of the base pair) and tilt (rotation about the short axis), with average values near 0° in ideal B-DNA but allowing flexibility for sequence-dependent bending. In alternative conformations, such as A-DNA, the axial rise shortens to about 0.28 nm with 11 base pairs per turn and a wider, shallower major groove, while Z-DNA features a left-handed helix with 12 base pairs per turn and an axial rise of approximately 0.37 nm, often stabilized in high-salt conditions or specific sequences like alternating purine-pyrimidines. These parameters enable the base pair to serve as a modular unit in diverse nucleic acid architectures.^[88]^[89]^[30] The consistent dimensions of base pairs facilitate practical applications in biophysics and genomics, such as estimating the physical length of genomes; for instance, the human haploid genome of approximately 3.2 billion base pairs extends to about 1.1 meters when linearized, assuming B-form geometry. Atomic force microscopy (AFM) leverages this scale for high-resolution imaging, achieving sub-nanometer precision to visualize individual base pairs or helical turns on surfaces, aiding in the study of DNA nanostructures and topology.^[90]^[91]^[92] In RNA, which typically adopts an A-form helix, the base pair exhibits a shorter axial rise of 0.28 nm and about 11 base pairs per turn, resulting in a more compact, elongated structure suited to functional folds like hairpins and ribozymes. This geometry influences RNA's role in designing synthetic nanostructures, where A-form parameters guide the assembly of RNA tiles or hybrid DNA-RNA scaffolds for precise molecular patterning.^[93] The evolutionary conservation of base pair spacing across domains of life underscores its fundamental role in nucleic acid architecture, enabling polymerases to translocate uniformly during replication and transcription regardless of sequence variation. This uniformity, preserved over billions of years, ensures compatibility with conserved enzymatic mechanisms.^[94]

Data Sources for Strengths

Experimental quantification of base pair interaction strengths in DNA and RNA relies on several biophysical techniques that probe thermodynamic stability and hydrogen bonding. Ultraviolet (UV) melting analysis measures melting temperature (Tm) curves by monitoring hyperchromicity at 260 nm as duplexes dissociate with increasing temperature, providing insights into overall stability influenced by base pairing.^[95] Differential scanning calorimetry (DSC) directly determines enthalpy (ΔH) and entropy (ΔS) changes during thermal denaturation by tracking heat capacity, revealing the energetic contributions of base pair formation and stacking.^[96] Nuclear magnetic resonance (NMR) spectroscopy assesses hydrogen bond strengths through chemical shifts of imino protons (typically 10-15 ppm for Watson-Crick pairs) and scalar couplings across H-bonds (e.g., ³J_{H-N} ≈ 1-2 Hz), offering atom-level resolution of pairing geometry and dynamics.^[97] Optical tweezers enable single-molecule force spectroscopy, rupturing individual base pairs at forces around 15 pN, which quantifies mechanical stability under tension.^[98] Key databases compile these experimental data into nearest-neighbor (NN) parameters for predictive modeling. The unified NN parameters for DNA, originally derived from UV melting and calorimetry data across oligonucleotides, polymers, and dumbbells, were established in 1998 and provide a standardized set for ΔG°_{37} calculations.^[99] These have been updated in the 2020s through expanded datasets like the Nearest Neighbor Database (NNDB), which incorporates additional DNA and RNA parameters, including modifications such as m⁶A, for improved accuracy in thermodynamic predictions.^[100] For RNA, RNAstructure software utilizes Turner rules, a comprehensive NN model based on optical melting experiments, to estimate helix stabilities.^[101] The DINAMelt server integrates these unified DNA parameters (SantaLucia) and RNA Turner rules to simulate melting profiles online, facilitating access to NN-based computations.^[102] Strength metrics from these sources emphasize free energy increments (ΔΔG°) for NN base pair steps, which capture both hydrogen bonding and stacking interactions. For DNA at 37°C and 1 M NaCl, an AT/TA pair contributes approximately -0.9 kcal/mol, while a GC/CG pair provides -2.2 kcal/mol, highlighting the greater stability of GC due to three hydrogen bonds versus two in AT.^[99] Similar values apply to RNA AU/UA (-0.9 kcal/mol) and GC/CG (-2.1 kcal/mol), with stacking matrices adjusting for sequence context in duplex formation. These parameters enable predictions of duplex free energies via summation: ΔG° = ΔG°_init + Σ ΔG°_NN + corrections. Recent updates from 2023-2025 include datasets on metal-mediated unnatural base pair (UBP) stability, where ions like Gd³⁺ coordinate 5-hydroxyuracil pairs, enhancing Tm by up to 26°C compared to canonical pairs, as measured by UV melting in synthetic duplexes.^[103] For RNA mismatches, molecular dynamics (MD) simulations have generated datasets revealing sequence-dependent dynamics, such as U:U wobble pairs exhibiting rapid opening-closing (lifetimes ~10-100 ns) flanked by stable helices, validated against NMR data. Despite their utility, these data sources exhibit limitations due to context-dependence, where NN parameters overlook tertiary interactions or long-range effects that can alter stabilities by 1-2 kcal/mol in complex structures. Additionally, in vitro measurements (e.g., high salt, 1 M NaCl) often overestimate duplex stability compared to in vivo conditions (crowded cellular environments, ~150 mM ions), necessitating adjusted "in vivo-like" parameters for better physiological predictions.^[104]

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Intercalators.1. Nature of stacking interactions between intercalators (ethidium, daunomycin, ellipticine, and 4′,6-diaminide-2-phenylindole) and DNA base pairs ...
[73]
Identification of Binding Mechanisms in Single Molecule–DNA ...
Daunomycin as an intercalant inserts into DNA via a stacking interaction of its aromatic ring system with the base pairs. Intercalated DNA should display a ...
[74]
Doxorubicin: Uses, Interactions, Mechanism of Action - DrugBank
It is generally thought to exert its antitumor effect by destabilizing DNA structures through intercalation, thus introducing DNA strand breakages and damages.Identification · Pharmacology · Interactions · References
[75]
Intercalating Agent - an overview | ScienceDirect Topics
Intercalating agents are molecules that insert between the bases in DNA, potentially causing frameshift ... toxic to proliferating cells. AI generated definition ...
[76]
Azidothymidine and other chain terminators are mutagenic for ... - NIH
Its therapeutic effects arise by its incorporation during HIV reverse transcription, resulting in chain termination. Azidothymidine is genotoxic, particularly ...
[77]
Doxorubicin induces an extensive transcriptional and metabolic ...
Sep 12, 2018 · Two different mechanisms, (i) intercalation of doxorubicin into DNA and inhibition of topoisomerase II leading to changes in chromatin structure ...
[78]
"Rule of thumb" for DNA volume per base pair - Generic - BioNumbers
"Rule of thumb" for DNA volume per base pair. Value, 1 nm^3. Organism, Generic. Reference, "Physical Biology of the Cell", Rob Phillips, Jane Kondev and Julie ...
[79]
[PDF] Lectures 2 & 3
In this type of DNA, the helical twist is 36o, meaning that there are 10.0 base pair/1 turn of helix.
[80]
[PDF] Nucleic Acid Structure - B. Stark
The six inter base pairs parameters (rise, twist, shift, roll, tilt, slide) describe the local conformation of a double helix at every base pair step. A table ...
[81]
The Human Genome - NCBI - NIH
The human genome, made of DNA, has two parts: a nuclear genome of about 3.2 billion nucleotides and a mitochondrial genome of 16,569 nucleotides.
[82]
High-Resolution Imaging of DNA Nanoarchitectures Using AFM
The diameter of a DNA molecule is about 2 nm. One whole revolution is completed after 10 base pairs, giving a pitch of 3.4 nm. The whole repeat is formed ...
[83]
Atomic force microscopy—A tool for structural and translational DNA ...
Atomic force microscopy (AFM) is a powerful imaging technique that allows for structural characterization of single biomolecules with nanoscale resolution.
[84]
Crystal structure of an Okazaki fragment at 2-A resolution
Aug 6, 2025 · We assume that the RNA-DNA hybrid duplex is the ideal A-form helix 6 , with 11 bp per turn of helix and 0.28 nm of rise per bp. The inter ...
[85]
Structure and function relationships in mammalian DNA polymerases
Accordingly, the fidelity of pol δ has evolved to be one of the highest among DNA polymerases, at a rate of ~ 1 base misinsertion per 105 nucleotides ...
[86]
Optical Melting Measurements of Nucleic Acid Thermodynamics - PMC
Optical melting experiments provide measurements of thermodynamic parameters for nucleic acids. These thermodynamic parameters are widely used in RNA structure ...Missing: DSC tweezers
[87]
Differential scanning calorimetry: An invaluable tool for a detailed ...
Differential Scanning Calorimetry (DSC) is a highly sensitive technique to study the thermotropic properties of many different biological macromolecules and ...
[88]
NMR scalar couplings across Watson–Crick base pair ... - PNAS
This paper describes the NMR observation of 15N—15N and 1H—15N scalar couplings across the hydrogen bonds in Watson–Crick base pairs in a DNA duplex ...
[89]
Single-molecule force spectroscopy: optical tweezers, magnetic ...
Conversely, the forces (~15 pN) and displacements (~nm) associated with nucleic acid folding are ideally suited for optical tweezers based measurements.
[90]
A unified view of polymer, dumbbell, and oligonucleotide DNA ...
The ΔH° and ΔS° parameters are analogously calculated from the parameters in Table 2 (22). The ΔG°37 can also be calculated from ΔH° and ΔS° parameters by using ...
[91]
NNDB: An Expanded Database of Nearest Neighbor Parameters for ...
Sep 1, 2024 · We expanded the database to include a set of DNA parameters and a set of RNA parameters that includes m6A in addition to the canonical RNA ...
[92]
Nearest Neighbor Database - Mathews Lab - University of Rochester
May 4, 2024 · These are the set of nearest neighbor parameters for DNA folding compiled by the Mathews group and released as part of RNAstructure package.
[93]
DINAMelt web server for nucleic acid melting prediction
Both Δ G and Δ H are computed using published nearest neighbor coefficients. We use the 'unified' parameters of SantaLucia ( 3 ) for DNA and the 'Turner lab' ...
[94]
Metal-mediated DNA strand displacement and molecular device ...
Aug 24, 2023 · We demonstrate the concept of metal-mediated base-pair switching to induce inter- and intramolecular DNA strand displacement in a metal-responsive manner.
[95]
In vivo-like nearest neighbor parameters improve prediction of ...
The in vivo-like adjustments have minimal effects on the prediction of RNA secondary structures determined in vitro and in silico, but markedly improve ...Missing: context | Show results with:context