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Nucleic acid tertiary structure
Nucleic acid tertiary structure
Nucleic acid tertiary structure
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Nucleic acid primary structureNucleic acid secondary structureNucleic acid double helixStem-loopPseudoknotNucleic acid quaternary structure
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Interactive image of nucleic acid structure (primary, secondary, tertiary, and quaternary) using DNA helices and examples from the VS ribozyme and telomerase and nucleosome. (PDB: ADNA, 1BNA, 4OCB, 4R4V, 1YMO, 1EQZ​)

Nucleic acid tertiary structure is the three-dimensional shape of a nucleic acid polymer.[1] RNA and DNA molecules are capable of diverse functions ranging from molecular recognition to catalysis. Such functions require a precise three-dimensional structure. While such structures are diverse and seemingly complex, they are composed of recurring, easily recognizable tertiary structural motifs that serve as molecular building blocks. Some of the most common motifs for RNA and DNA tertiary structure are described below, but this information is based on a limited number of solved structures. Many more tertiary structural motifs will be revealed as new RNA and DNA molecules are structurally characterized.

Helical structures

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The structures of the A-, B-, and Z-DNA double helix structures.

Double helix

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Main article: Nucleic acid double helix

The double helix is the dominant tertiary structure for biological DNA, and is also a possible structure for RNA. Three DNA conformations are believed to be found in nature, A-DNA, B-DNA, and Z-DNA. The "B" form described by James D. Watson and Francis Crick is believed to predominate in cells.[2] James D. Watson and Francis Crick described this structure as a double helix with a radius of 10 Å and pitch of 34 Å, making one complete turn about its axis every 10 bp of sequence.[3] The double helix makes one complete turn about its axis every 10.4–10.5 base pairs in solution. This frequency of twist (known as the helical pitch) depends largely on stacking forces that each base exerts on its neighbours in the chain. Double-helical RNA adopts a conformation similar to the A-form structure.

Other conformations are possible; in fact, only the letters F, Q, U, V, and Y are now available to describe any new DNA structure that may appear in the future.[4][5] However, most of these forms have been created synthetically and have not been observed in naturally occurring biological systems.

RNA triplexes
Major groove triples in the group II intron in Oceanobacillus Iheyensis. Each stacked layer is formed by one triplex with a different color scheme. Hydrogen bonds between triplexes are shown in black dashed lines. "N" atoms are colored in blue and "O" atoms in red. From top to bottom, the residues on the left side are G288, C289, and C377.[6]
Close-up rendering of the U114:A175-U101 major groove (Hoogsteen base) triplex formed within the wild type pseudoknot of Human Telomerase RNA. Hydrogen bonds are shown in black dashed lines. "N" atoms are colored in blue and "o" atoms in red.[7]

Major and minor groove triplexes

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The minor groove triplex is a ubiquitous RNA structural motif. Because interactions with the minor groove are often mediated by the 2'-OH of the ribose sugar, this RNA motif looks very different from its DNA equivalent. The most common example of a minor groove triple is the A-minor motif, or the insertion of adenosine bases into the minor groove (see above). However, this motif is not restricted to adenosines, as other nucleobases have also been observed to interact with the RNA minor groove.

The minor groove presents a near-perfect complement for an inserted base. This allows for optimal van der Waals contacts, extensive hydrogen bonding and hydrophobic surface burial, and creates a highly energetically favorable interaction.[8][9] Because minor groove triples are capable of stably packing a free loop and helix, they are key elements in the structure of large ribonucleotides, including the group I intron,[10] the group II intron,[11] and the ribosome.

Quadruplexes
Above:Typical Ring Structure of a Hoogsteen paired G-quartet.[12]
Above: Quadruplex seen in crystal structure of Malachite Green RNA aptamer. G29 involved in major groove, minor groove, and Watson-Crick hydrogen-bonding with three other bases.[13]

Although the major groove of standard A-form RNA is fairly narrow and therefore less available for triplex interaction than the minor groove, major groove triplex interactions can be observed in several RNA structures. These structures consist of several combinations of base pair and Hoogsteen interactions. For example, the GGC triplex (GGC amino(N-2)-N-7, imino-carbonyl, carbonyl-amino(N-4); Watson-Crick) observed in the 50S ribosome, composed of a Watson-Crick type G-C pair and an incoming G which forms a pseudo-Hoogsteen network of hydrogen bonding interactions between both bases involved in the canonical pairing.[12] Other notable examples of major groove triplexes include (i) the catalytic core of the group II intron shown in the figure at left [6] (ii) a catalytically essential triple helix observed in human telomerase RNA[7] (iii) the SAM-II riboswitch[14] and (iv) the element for nuclear expression (ENE), which acts as an RNA stabilization element through triple helix formation with the poly(A) tail.[15][16]

Triple-stranded DNA is also possible from Hoogsteen or reversed Hoogsteen hydrogen bonds in the major groove of B-form DNA.

Quadruplexes

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Besides double helices and the above-mentioned triplexes, RNA and DNA can both also form quadruple helices. There are diverse structures of RNA base quadruplexes. Four consecutive guanine residues can form a quadruplex in RNA by Hoogsteen hydrogen bonds to form a “Hoogsteen ring” (See Figure).[12] G-C and A-U pairs can also form base quadruplex with a combination of Watson-Crick pairing and noncanonical pairing in the minor groove.[17]

The core of malachite green aptamer is also a kind of base quadruplex with a different hydrogen bonding pattern (See Figure).[13] The quadruplex can repeat several times consecutively, producing an immensely stable structure.

The unique structure of quadruplex regions in RNA may serve different functions in a biological system. Two important functions are the binding potential with ligands or proteins, and its ability to stabilize the whole tertiary structure of DNA or RNA. The strong structure can inhibit or modulate transcription and replication, such as in the telomeres of chromosomes and the UTR of mRNA.[18] The base identity is important towards ligand binding. The G-quartet typically binds monovalent cations such as potassium, while other bases can bind numerous other ligands such as hypoxanthine in a U-U-C-U quadruplex.[17]

Along with these functions, the G-quadruplex in the mRNA around the ribosome binding regions could serve as a regulator of gene expression in bacteria.[19] There may be more interesting structures and functions yet to be discovered in vivo.

Coaxial stacking

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Secondary (inset) and tertiary structure of tRNA demonstrating coaxial stacking.[20]

Coaxial stacking, otherwise known as helical stacking, is a major determinant of higher order RNA tertiary structure. Coaxial stacking occurs when two RNA duplexes form a contiguous helix, which is stabilized by base stacking at the interface of the two helices. Coaxial stacking was noted in the crystal structure of tRNAPhe.[21] More recently, coaxial stacking has been observed in higher order structures of many ribozymes, including many forms of the self-splicing group I and group II introns. Common coaxial stacking motifs include the kissing loop interaction and the pseudoknot. The stability of these interactions can be predicted by an adaptation of “Turner’s rules”.[22][23]

In 1994, Walter and Turner determined the free energy contributions of nearest neighbor stacking interactions within a helix-helix interface by using a model system that created a helix-helix interface between a short oligomer and a four-nucleotide overhang at the end of a hairpin stem . Their experiments confirmed that the thermodynamic contribution of base-stacking between two helical secondary structures closely mimics the thermodynamics of standard duplex formation (nearest neighbor interactions predict the thermodynamic stability of the resulting helix). The relative stability of nearest neighbor interactions can be used to predict favorable coaxial stacking based on known secondary structure. Walter and Turner found that, on average, prediction of RNA structure improved from 67% to 74% accuracy when coaxial stacking contributions were included.[24]

Most well-studied RNA tertiary structures contain examples of coaxial stacking. Some prominent examples are tRNA-Phe, group I introns, group II introns, and ribosomal RNAs. Crystal structures of tRNA revealed the presence of two extended helices that result from coaxial stacking of the amino-acid acceptor stem with the T-arm, and stacking of the D- and anticodon-arms. These interactions within tRNA orient the anticodon stem perpendicularly to the amino-acid stem, leading to the functional L-shaped tertiary structure.[21] In group I introns, the P4 and P6 helices were shown to coaxially stack using a combination of biochemical[25] and crystallographic methods. The P456 crystal structure provided a detailed view of how coaxial stacking stabilizes the packing of RNA helices into tertiary structures.[26] In the self-splicing group II intron from Oceanobacillus iheyensis, the IA and IB stems coaxially stack and contribute to the relative orientation of the constituent helices of a five-way junction.[6] This orientation facilitates proper folding of the active site of the functional ribozyme. The ribosome contains numerous examples of coaxial stacking, including stacked segments as long as 70 bp.[27]

formation of a pseudoknot with coaxial stacking of the two helices

Two common motifs involving coaxial stacking are kissing loops and pseudoknots. In kissing loop interactions, the single-stranded loop regions of two hairpins interact through base pairing, forming a composite, coaxially stacked helix. Notably, this structure allows all of the nucleotides in each loop to participate in base-pairing and stacking interactions. This motif was visualized and studied using NMR analysis by Lee and Crothers.[28] The pseudoknot motif occurs when a single stranded region of a hairpin loop base-pairs with an upstream or downstream sequence within the same RNA strand. The two resulting duplex regions often stack upon one another, forming a stable coaxially stacked composite helix. One example of a pseudoknot motif is the highly stable Hepatitis Delta virus ribozyme, in which the backbone shows an overall double pseudoknot topology.[29]

An effect similar to coaxial stacking has been observed in rationally designed DNA structures. DNA origami structures contain a large number of double helixes with exposed blunt ends. These structures were observed to stick together along the edges that contained these exposed blunt ends, due to the hydrophobic stacking interactions.[30] By combining these rationally designed DNA nanostructures and DNA-PAINT super-resolution imaging, researchers discerned individual strength of stacking energies between all possible dinucleotides.[31]

Measurement of coaxial stacking in nucleic acid

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Early measurements of coaxial stacking were performed using biochemical assays that studies the relative migration of different nucleic acid molecules based on their conformation and the kind of interactions present. Short DNA molecules containing nicks that could still stack coaxially migrated faster than DNA molecules containing gaps and thus had no coaxial stacking. This could be explained by polymeric properties of DNA where are more rigid rod like molecule will migrate faster along an electrical gradient in a matrix compared to a more flexible molecule.[32] Development of newer techniques such as optical tweezers and the ability to fold DNA nanostructures led to measurement so of DNA bundles and their ability to stack with each other. The force needed to pull these bundles apart using optical tweezers could then be analyzed to measure the base-pair stacking energies.[33] These measurements were performed mainly under non-equilibrium conditions and various extrapolations were made to arrive at the exact values of coaxial stacking between bases. Recent single-molecule studies using DNA nanostructures and DNA-PAINT super-resolution microscopy has allowed for measurement of these interaction between dinucleotides using in-depth kinetic analysis of binding times of short DNA molecules to their complimentary sequences in the presence or absence of DNA-stacking interactions.[31]

Other motifs

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Tetraloop-receptor interactions

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Stick representation of a GAAA tetraloop - an example from the GNRA tetraloop family.[34]

Tetraloop-receptor interactions combine base-pairing and stacking interactions between the loop nucleotides of a tetraloop motif and a receptor motif located within an RNA duplex, creating a tertiary contact that stabilizes the global tertiary fold of an RNA molecule. Tetraloops are also possible structures in DNA duplexes.[35]

Stem-loops can vary greatly in size and sequence, but tetraloops of four nucleotides are very common and they usually belong to one of three categories, based on sequence.[36] These three families are the CUYG, UNCG, and GNRA (see figure on the right) tetraloops.[37] In each of these tetraloop families, the second and third nucleotides form a turn in the RNA strand and a base-pair between the first and fourth nucleotides stabilizes the stemloop structure. It has been determined, in general, that the stability of the tetraloop depends on the composition of bases within the loop and on the composition of this "closing base pair".[38] The GNRA family of tetraloops is the most commonly observed within Tetraloop-receptor interactions. Additionally, the UMAC tetraloops are known to be alternative versions of the GNRA loops, both sharing similar backbone structures; despite the similarities, they differ in the possible long-range interactions they are capable of.[39]

GAAA Tetraloop and Receptor: Stick representation of tetraloop (yellow) and its receptor, showing both Watson-Crick and Hoogsteen base-pairing.[34]

“Tetraloop receptor motifs” are long-range tertiary interactions[40] consisting of hydrogen bonding between the bases in the tetraloop to stemloop sequences in distal sections of the secondary RNA structure.[41] In addition to hydrogen bonding, stacking interactions are an important component of these tertiary interactions. For example, in GNRA-tetraloop interactions, the second nucleotide of the tetraloop stacks directly on an A-platform motif (see above) within the receptor.[26] The sequence of the tetraloop and its receptor often covary so that the same type of tertiary contact can be made with different isoforms of the tetraloop and its cognate receptor.[42]

For example, the self-splicing group I intron relies on tetraloop receptor motifs for its structure and function.[26][41] Specifically, the three adenine residues of the canonical GAAA motif stack on top of the receptor helix and form multiple stabilizing hydrogen bonds with the receptor. The first adenine of the GAAA sequence forms a triple base-pair with the receptor AU bases. The second adenine is stabilized by hydrogen bonds with the same uridine, as well as via its 2'-OH with the receptor and via interactions with the guanine of the GAAA tetraloop. The third adenine forms a triple base pair.

A-minor motif

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A-minor Interactions
Type I A-minor interaction: Type I interactions are the most common, strongest A-minor interactions, as they involve numerous hydrogen bonds, and bury the incoming A base in the minor groove.[43]
Type II A-minor interaction: Type II interactions involve the 2'-OH group and N3 of the adenosine. The adenosine interacts with the cytosine's 2'-OH group in the minor groove. The strength of this interaction is on the order of the Type I interaction.[43]

The A-minor motif is a ubiquitous RNA tertiary structural motif. It is formed by the insertion of an unpaired nucleoside into the minor groove of an RNA duplex. As such it is an example of a minor groove triple. Although guanosine, cytosine and uridine can also form minor groove triple interactions, minor groove interactions by adenine are very common. In the case of adenine, the N1-C2-N3 edge of the inserting base forms hydrogen bonds with one or both of the 2’-OH's of the duplex, as well as the bases of the duplex (see figure: A-minor interactions). The host duplex is often a G-C basepair.

A-minor motifs have been separated into four classes,[8][9] types 0 to III, based upon the position of the inserting base relative to the two 2’-OH's of the Watson-Crick base pair. In type I and II A-minor motifs, N3 of adenine is inserted deeply within the minor groove of the duplex (see figure: A minor interactions - type II interaction), and there is good shape complementarity with the base pair. Unlike types 0 and III, type I and II interactions are specific for adenine due to hydrogen bonding interactions. In the type III interaction, both the O2' and N3 of the inserting base are associated less closely with the minor groove of the duplex. Type 0 and III motifs are weaker and non-specific because they are mediated by interactions with a single 2’-OH (see figure: A-minor Interactions - type 0 and type III interactions).

The A-minor motif is among the most common RNA structural motifs in the ribosome, where it contributes to the binding of tRNA to the 23S subunit.[44] They most often stabilize RNA duplex interactions in loops and helices, such as in the core of group II introns.[6]

An interesting example of A-minor is its role in anticodon recognition. The ribosome must discriminate between correct and incorrect codon-anticodon pairs. It does so, in part, through the insertion of adenine bases into the minor groove. Incorrect codon-anticodon pairs will present distorted helical geometry, which will prevent the A-minor interaction from stabilizing the binding, and increase the dissociation rate of the incorrect tRNA.[45]

An analysis of A-minor motifs in the 23S ribosomal RNA has revealed a hierarchical network of structural dependencies, suggested to be related to ribosomal evolution and to the order of events that led to the development of the modern bacterial large subunit.[46]

The A-minor motif and it's novel subclass, WC/H A-minor interactions, are reported to fortify other RNA tertiary structures such as major groove triple helices identified in RNA stabilization elements.[16][15]

Ribose zipper

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Ribose Zippers: View of a canonical ribose zipper between two RNA backbones.[34]

The ribose zipper is an RNA tertiary structural element in which two RNA chains are held together by hydrogen bonding interactions involving the 2’OH of ribose sugars on different strands. The 2'OH can behave as both hydrogen bond donor and acceptor, which allows formation of bifurcated hydrogen bonds with another 2’ OH.[47][48]

Numerous forms of ribose zipper have been reported, but a common type involves four hydrogen bonds between 2'-OH groups of two adjacent sugars. Ribose zippers commonly occur in arrays that stabilize interactions between separate RNA strands.[49] Ribose zippers are often observed as Stem-loop interactions with very low sequence specificity. However, in the small and large ribosomal subunits, there exists a propensity for ribose zippers of the CC/AA sequence- two cytosines on the first chain paired to two adenines on the second chain.

Role of metal ions

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Metal Ion Binding in the Group I Intron
PDB rendering of Group I intron inner sphere magnesium coordination. The two red balls indicate magnesium ions and dashed lines coming from the ions indicate coordination with the respective groups on nucleotides. The color-coding scheme is as follows: green=carbon, orange=phosphate, pink=oxygen, blue=nitrogen.[50]
PDB rendering of Group 1 intron P5c binding pocket demonstrating outer sphere coordination. Here, the six amines of osmium hexamine(III) fulfill the role generally served by water molecules and mediate the ion’s interaction with the major groove. Coordination via hydrogen bonds is indicated by dashed lines and osmium is rendered in pink, all other colors are as above.[34]

Functional RNAs are often folded, stable molecules with three-dimensional shapes rather than floppy, linear strands.[51] Cations are essential for thermodynamic stabilization of RNA tertiary structures. Metal cations that bind RNA can be monovalent, divalent or trivalent. Potassium (K+) is a common monovalent ion that binds RNA. A common divalent ion that binds RNA is magnesium (Mg2+). Other ions including sodium (Na+), calcium (Ca2+) and manganese (Mn2+) have been found to bind RNA in vivo and in vitro. Multivalent organic cations such as spermidine or spermine are also found in cells and these make important contributions to RNA folding. Trivalent ions such as cobalt hexamine or lanthanide ions such as terbium (Tb3+) are useful experimental tools for studying metal binding to RNA.[52][53]

A metal ion can interact with RNA in multiple ways. An ion can associate diffusely with the RNA backbone, shielding otherwise unfavorable electrostatic interactions. This charge screening is often fulfilled by monovalent ions. Site-bound ions stabilize specific elements of RNA tertiary structure. Site-bound interactions can be further subdivided into two categories depending on whether water mediates the metal binding. “Outer sphere” interactions are mediated by water molecules that surround the metal ion. For example, magnesium hexahydrate interacts with and stabilizes specific RNA tertiary structure motifs via interactions with guanosine in the major groove. Conversely, “inner sphere” interactions are directly mediated by the metal ion. RNA often folds in multiple stages and these steps can be stabilized by different types of cations. In the early stages, RNA forms secondary structures stabilized through the binding of monovalent cations, divalent cations and polyanionic amines in order to neutralize the polyanionic backbone. The later stages of this process involve the formation of RNA tertiary structure, which is stabilized almost largely through the binding of divalent ions such as magnesium with possible contributions from potassium binding.

Metal-binding sites are often localized in the deep and narrow major groove of the RNA duplex, coordinating to the Hoogsteen edges of purines. In particular, metal cations stabilize sites of backbone twisting where tight packing of phosphates results in a region of dense negative charge. There are several metal ion-binding motifs in RNA duplexes that have been identified in crystal structures. For instance, in the P4-P6 domain of the Tetrahymena thermophila group I intron, several ion-binding sites consist of tandem G-U wobble pairs and tandem G-A mismatches, in which divalent cations interact with the Hoogsteen edge of guanosine via O6 and N7.[54][55][56] Another ion-binding motif in the Tetrahymena group I intron is the A-A platform motif, in which consecutive adenosines in the same strand of RNA form a non-canonical pseudobase pair.[57] Unlike the tandem G-U motif, the A-A platform motif binds preferentially to monovalent cations. In many of these motifs, absence of the monovalent or divalent cations results in either greater flexibility or loss of tertiary structure.

Divalent metal ions, especially magnesium, have been found to be important for the structure of DNA junctions such as the Holliday junction intermediate in genetic recombination. The magnesium ion shields the negatively charged phosphate groups in the junction and allows them to be positioned closer together, allowing a stacked conformation rather than an unstacked conformation.[58] Magnesium is vital in stabilizing these kinds of junctions in artificially designed structures used in DNA nanotechnology, such as the double crossover motif.[59]

History

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Main article: History of molecular biology

The earliest work in RNA structural biology coincided, more or less, with the work being done on DNA in the early 1950s. In their seminal 1953 paper, Watson and Crick suggested that van der Waals crowding by the 2`OH group of ribose would preclude RNA from adopting a double helical structure identical to the model they proposed - what we now know as B-form DNA.[60] This provoked questions about the three dimensional structure of RNA: could this molecule form some type of helical structure, and if so, how?

In the mid-1960s, the role of tRNA in protein synthesis was being intensively studied. In 1965, Holley et al. purified and sequenced the first tRNA molecule, initially proposing that it adopted a cloverleaf structure, based largely on the ability of certain regions of the molecule to form stem loop structures.[61] The isolation of tRNA proved to be the first major windfall in RNA structural biology. In 1971, Kim et al. achieved another breakthrough, producing crystals of yeast tRNAPHE that diffracted to 2-3 Ångström resolutions by using spermine, a naturally occurring polyamine, which bound to and stabilized the tRNA.[62]

For a considerable time following the first tRNA structures, the field of RNA structure did not dramatically advance. The ability to study an RNA structure depended upon the potential to isolate the RNA target. This proved limiting to the field for many years, in part because other known targets - i.e., the ribosome - were significantly more difficult to isolate and crystallize. As such, for some twenty years following the original publication of the tRNAPHE structure, the structures of only a handful of other RNA targets were solved, with almost all of these belonging to the transfer RNA family.[63]

This unfortunate lack of scope would eventually be overcome largely because of two major advancements in nucleic acid research: the identification of ribozymes, and the ability to produce them via in vitro transcription. Subsequent to Tom Cech's publication implicating the Tetrahymena group I intron as an autocatalytic ribozyme,[64] and Sidney Altman's report of catalysis by ribonuclease P RNA,[65] several other catalytic RNAs were identified in the late 1980s,[66] including the hammerhead ribozyme. In 1994, McKay et al. published the structure of a 'hammerhead RNA-DNA ribozyme-inhibitor complex' at 2.6 Ångström resolution, in which the autocatalytic activity of the ribozyme was disrupted via binding to a DNA substrate.[67] In addition to the advances being made in global structure determination via crystallography, the early 1990s also saw the implementation of NMR as a powerful technique in RNA structural biology. Investigations such as this enabled a more precise characterization of the base pairing and base stacking interactions which stabilized the global folds of large RNA molecules.

The resurgence of RNA structural biology in the mid-1990s has caused a veritable explosion in the field of nucleic acid structural research. Since the publication of the hammerhead and P4-6 structures, numerous major contributions to the field have been made. Some of the most noteworthy examples include the structures of the Group I and Group II introns,[6] and the Ribosome.[43] The first three structures were produced using in vitro transcription, and that NMR has played a role in investigating partial components of all four structures - testaments to the indispensability of both techniques for RNA research. The 2009 Nobel Prize in Chemistry was awarded to Ada Yonath, Venkatraman Ramakrishnan, and Thomas Steitz for their structural work on the ribosome, demonstrating the prominent role RNA structural biology has taken in modern molecular biology.

See also

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References

[edit]
Revisions and contributorsEdit on WikipediaRead on Wikipedia

Nucleic acid tertiary structure

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from Grokipedia
Nucleic acid tertiary structure refers to the three-dimensional folding of a nucleic acid chain achieved through long-range interactions between secondary structural elements, such as base stacking, hydrogen bonding, and electrostatic contacts, resulting in a compact and stable conformation essential for biological function. This level of organization builds upon the primary sequence of nucleotides and the secondary structures like double helices in DNA or stem-loops in RNA, enabling diverse roles from genetic storage to enzymatic activity. For DNA, tertiary structure primarily manifests as supercoiling of the double helix, where the linear or circular molecule twists upon itself to achieve a more compact form, facilitating storage within the cell nucleus. This supercoiling can be positive or negative, with negative supercoils aiding in unwinding the helix for processes like replication and transcription; enzymes such as topoisomerases and DNA gyrase regulate these twists to maintain appropriate tension. Higher-order packaging involves wrapping around histone proteins to form nucleosomes, which organize into chromatin and chromosomes, allowing compaction of over 2 meters of DNA in a typical mammalian cell nucleus. In RNA, tertiary structure is more varied and intricate, often stabilized by multivalent cations that neutralize the negatively charged phosphate backbone, with assembly of multiple helical segments into globular architectures through motifs like coaxial stacking, where adjacent helices align end-to-end, and pseudoknots, formed by base pairing between loop and stem regions. Examples include the L-shaped fold of (tRNA), stabilized by interactions between its D-arm and T-arm helices, or the catalytic core of ribozymes like the ribosome's peptidyl transferase center. These structures enable RNA to perform regulatory, catalytic, and scaffolding roles, with dynamics allowing conformational changes in response to ligands or cellular conditions; chemical probing methods reveal these folds by assessing reactivity in solution. Overall, nucleic acid tertiary structures are dynamically regulated and critical for cellular processes, with disruptions linked to diseases such as cancer or genetic disorders; ongoing research uses computational modeling and experimental techniques to predict and visualize these folds for therapeutic applications.

Fundamentals

Definition and overview

Nucleic acid tertiary structure refers to the three-dimensional spatial arrangement of secondary structural elements within a single DNA or RNA molecule, stabilized by long-range interactions including hydrogen bonds, electrostatic forces, and hydrophobic base stacking. These elements, such as helices and loops, fold together to form a compact global architecture that is crucial for the molecule's stability and function. The basic building blocks are nucleotides—each consisting of a nitrogenous base, a sugar (deoxyribose in DNA or ribose in RNA), and a phosphate group—connected via phosphodiester linkages in the backbone, with π-π stacking between adjacent bases providing essential hydrophobic stabilization for folding. Unlike secondary structure, which emerges from local, sequential base pairing (e.g., Watson-Crick pairs forming double-helical stems in RNA or the canonical double helix in DNA), tertiary structure involves non-local contacts that bring distant regions of the chain into proximity, creating higher-order motifs. This level of organization builds upon the primary structure, the linear sequence of nucleotides, which encodes the information necessary to guide these interactions but does not directly dictate the final 3D form. Tertiary folding is vital for function, enabling processes such as and . In , ribozymes like the group I achieve self-splicing through precise tertiary contacts that align substrates at the . Riboswitches, such as the adenine-binding variant, undergo ligand-induced tertiary rearrangements to toggle by altering access to transcription termination sites. Similarly, in DNA, tertiary structures including supercoiling of and G-quadruplexes in telomeric and promoter regions facilitate regulatory roles by influencing protein binding and transcriptional control.

Relation to primary and secondary structures

The tertiary structure of nucleic acids arises hierarchically from the primary sequence of nucleotides, which encodes the potential for local base-pairing interactions that form secondary structural elements, such as helices and loops in RNA or double-stranded regions in DNA; these secondary motifs then assemble into the global three-dimensional fold through long-range tertiary contacts. This model parallels the structural organization in proteins but is adapted to the unique chemical properties of nucleic acids, where the primary sequence directly influences secondary structure stability via Watson-Crick base pairing, providing scaffolds for tertiary interactions like coaxial stacking or groove binding. In both RNA and DNA, the primary-to-secondary transition is largely deterministic, governed by sequence complementarity, while tertiary folding introduces greater variability dependent on environmental factors. Folding principles in nucleic acids emphasize this hierarchy, particularly in RNA, where secondary structures form rapidly as stable intermediates, creating a that guides slower tertiary assembly and reduces conformational . For instance, in ribosomal RNA domains, base-paired helices emerge first, followed by docking of loops to form the compact tertiary core. In DNA, folding to tertiary structures, such as in promoter regions or G-quadruplexes, often proceeds cooperatively, with secondary pairing and tertiary motifs stabilizing each other in a concerted manner rather than strictly sequential steps. This cooperative aspect in DNA contrasts with RNA's more modular hierarchy, reflecting DNA's tendency toward rigid double-helical scaffolds that bend or twist into higher-order forms. The driving force for these transitions is free energy minimization, with contributions from hydrogen bonding that stabilizes canonical base pairs in secondary elements, π-π stacking interactions that enhance helical rigidity and alignments in tertiary domains, and effects that favor the burial of hydrophobic bases away from aqueous environments. In , stacking energies can contribute up to 0.5-3 kcal/mol per base step, while hydrogen bonds provide specificity; penalties are offset by coordination in the grooves. Tertiary folding further lowers the overall free energy by integrating these forces across distant sequence regions, creating a funnel-shaped that channels the molecule toward the native state. This resolves an adaptation of to nucleic acids, where the immense number of possible conformations (e.g., ~3^n for an n-nucleotide ) is navigated efficiently through hierarchical barriers that limit exploration to viable secondary scaffolds, enabling folding on biologically relevant timescales. Secondary structural irregularities, such as mismatches or bulges, often initiate transitions to tertiary distortions by introducing flexibility or recognition sites that propagate conformational changes to the global fold. In RNA, a single unpaired nucleotide in a helix (bulge) can distort the A-form geometry, facilitating tertiary contacts like loop-receptor binding in ribozymes. Similarly, in DNA, mismatches in promoter sequences can bend the duplex, promoting tertiary looping essential for regulatory protein binding. These elements underscore how primary sequence variations at the secondary level fine-tune tertiary architecture for functional diversity.

Helical and Multistranded Structures

Double helix variants

The canonical B-form double helix of DNA, first described by Watson and Crick, serves as the primary scaffold for nucleic acid tertiary structures under physiological conditions. It features a right-handed helix with approximately 10.5 base pairs per turn, an axial rise of 3.4 Å per base pair, a helical pitch of 34 Å, and a diameter of about 20 Å. The major groove is wide (12 Å) and deep, while the minor groove is narrow (6 Å) and deep, facilitating specific interactions with proteins that recognize sequence-dependent distortions in tertiary folding, such as bending or unwinding in chromatin loops.90243-4) A-DNA represents a variant adopted under dehydrating conditions, such as in fiber preparations or certain crystal environments, exhibiting a shorter and wider right-handed compared to B-DNA. Key parameters include 11 base pairs per turn, an axial rise of 2.6 , a pitch of 28 , and a of 23 , with a shallow major groove (2.7 wide) and a deep minor groove (11 wide). This conformation, resembling double-stranded structures, arises from C3'-endo sugar puckering and is triggered by low humidity, promoting base stacking that influences tertiary motifs like coaxial stacking in folding or DNA-RNA hybrids during transcription. In tertiary contexts, A-form segments enable groove-specific protein binding and contribute to helical distortions that stabilize larger folds, such as in complexes.90243-4) Z-DNA, a left-handed helix, forms in alternating purine-pyrimidine sequences, particularly GC-rich regions, under high salt concentrations or negative supercoiling. It has 12 base pairs per turn, an axial rise of 3.8 Å, a pitch of 45 Å, and a narrow diameter of 18 Å, with a nearly flat major groove and a deep, narrow minor groove. The zigzag phosphate backbone and syn glycosidic conformation for purines distinguish it from right-handed forms, often stabilized by proteins like ADAR1. In tertiary structures, Z-DNA facilitates sharp bends (up to 11°) at B-Z junctions and participates in chromatin looping or gene regulation by altering helical writhe, as observed in X-ray crystallography of junctions where axes displace by 5.2 Å to accommodate folding. Helical parameters like twist (–30°), roll, and tilt, measured via X-ray diffraction, reveal sequence-induced variations that propagate distortions for long-range tertiary interactions.
ParameterB-DNAA-DNAZ-DNA
HandednessRight-handedRight-handedLeft-handed
Base pairs/turn10.51112
Axial rise (Å/bp)3.42.63.8
Helix pitch (Å)342845
Helix diameter (Å)202318
Major groove width (Å)122.7~2 (flat)
Minor groove width (Å)611~2 (deep)
Twist angle (°)+36+33–30
These variants, characterized through , underscore how environmental triggers and sequence composition modulate helical geometry to support tertiary folding without disrupting Watson-Crick base pairing.90243-4)

Triplex structures

Triplex structures in nucleic acids involve the association of a third strand with a double helix, forming a three-stranded helical motif that contributes to higher-order folding and functional . These structures typically arise in sequences with polypurine/polypyrimidine tracts, where the third strand binds via Hoogsteen or reverse Hoogsteen hydrogen bonding, distinct from the Watson-Crick pairing in the duplex core. In DNA, triplex formation often occurs in the major groove, while RNA-involving triplexes can engage either the major or minor groove depending on the strand orientation and composition. H-DNA represents an intramolecular triplex in supercoiled DNA, where a single strand folds back to invade the duplex in homopurine/homopyrimidine mirror repeats, displacing the pyrimidine-rich strand as a single-stranded loop. The third strand, typically pyrimidine-rich, binds parallel to the purine strand in the major groove through Hoogsteen pairing, forming triplets such as T-AT and protonated C-GC⁺. This structure was first identified in superhelical plasmids, marking it as the earliest discovered multistranded DNA conformation. H-DNA formation is promoted by negative supercoiling, which provides the torsional stress to unwind the duplex, and is favored in sequences like (GA/TC)n or (GAA/TTC)n repeats. Intermolecular triplexes extend this motif to separate strands, including DNA-DNA or -DNA hybrids. In -DNA triplexes, the third strand often binds antiparallel to the DNA strand in the minor groove via reverse Hoogsteen pairing, forming stable triplets like rU-AT or rC⁺-GC, particularly in GA-rich motifs. These structures can also form in the major groove with parallel Hoogsteen geometry, as seen in long noncoding RNAs targeting promoter regions. triplexes among all- strands similarly rely on Hoogsteen interactions but are less common . Stability of triplexes is highly sequence- and condition-dependent, with polypurine/polypyrimidine tracts enabling selective third-strand invasion. The C-GC⁺ triplet requires cytosine protonation, making formation pH-sensitive and optimal at acidic conditions (pH < 6), while T-AT triplets are more neutral-pH stable. Superhelical density, ionic strength, and molecular crowding further enhance persistence, with triplexes resisting enzymatic digestion like DNase I in chromatin contexts. In tertiary architecture, triplexes play key roles in and . H-DNA in polypurine/pyrimidine tracts acts as a , facilitating transcription initiation by altering local topology in promoters and contributing to replication pausing or termination. In , expanded CGG repeats form H-DNA-like triplexes that promote repeat instability and methylation spreading, silencing the gene. RNA-DNA triplexes recruit regulatory complexes, such as PRC2 for epigenetic silencing or p300 for activation, influencing in development and stress responses. Additionally, triplex motifs in telomeric regions support end-protection and maintenance by stabilizing non-canonical folds during replication.

Quadruplex structures

G-quadruplexes, often abbreviated as G4s, are non-canonical four-stranded nucleic acid structures that form in guanine-rich sequences of DNA and RNA. These structures are built from stacks of two or more G-tetrads, where each tetrad consists of four guanine bases arranged in a planar configuration and interconnected through Hoogsteen hydrogen bonding. The Hoogsteen pairing involves the N7 and C6 atoms of guanine, differing from the Watson-Crick pairing in double helices. G4 formation is promoted under physiological conditions, particularly in the presence of monovalent cations such as potassium (K⁺), which coordinate between the O6 carbonyl oxygens of adjacent guanines in the tetrad core, enhancing stability through electrostatic shielding of the negatively charged phosphate backbone. Sodium (Na⁺) can also stabilize G4s, though K⁺ provides superior coordination due to its optimal ionic radius. G4 topologies vary based on strand orientation and loop configurations, leading to parallel, antiparallel, or hybrid arrangements. In parallel topologies, all strands run in the same 5'-to-3' direction, often featuring propeller loops that connect successive tetrads. Antiparallel structures include diagonal or bulge loops, where strands alternate direction, resulting in more compact folds. These variations influence the groove widths and overall architecture, with parallel G4s typically exhibiting narrower grooves compared to antiparallel ones. The number of stacked tetrads (usually two to four) and intervening loops further diversifies G4 conformations, as observed in high-resolution NMR and studies. RNA G-quadruplexes generally exhibit greater thermodynamic stability than their DNA counterparts, attributed to the 2'-OH group on , which enables additional hydrogen bonding and reduces flexibility in the sugar-phosphate backbone. For instance, G4s often form more readily and resist unfolding at higher temperatures, with melting temperatures up to 10-20°C higher than equivalent DNA sequences under similar ionic conditions. This enhanced stability in arises from denser hydration shells and more efficient stacking interactions between tetrads. In biological contexts, are prevalent in telomeres, where they form on the G-rich strand and regulate telomere maintenance by inhibiting activity during replication. They also occur in gene promoters, such as the c-MYC , where a G4 in the nuclease hypersensitivity element represses transcription by impeding progression. In mRNA 5' untranslated regions (UTRs), G4s typically inhibit initiation by blocking scanning, as exemplified in the NRAS proto-oncogene where proximity to the 5' enhances this repressive effect. These roles extend to broader functions in replication stalling and , with G4 resolution often requiring specialized helicases. The complementary C-rich strand to G4-forming sequences can adopt i-motif structures, which are four-stranded intercalated cytosine quadruplexes stabilized at slightly acidic through hemiprotonated C-C⁺ base pairs. Unlike , i-motifs feature two parallel duplexes zipped together, with stability enhanced by intercalation and hydrophobic interactions, and they often coexist or compete with in duplex regions like telomeres and promoters.

Stacking Interactions

Coaxial stacking

Coaxial stacking refers to the alignment of base pairs from the ends of two adjacent helical segments along a shared helical axis, effectively extending the continuity of the in tertiary structures. This tertiary interaction typically occurs at junctions in , such as multibranch loops or exterior loops, where the helices are connected either directly (flush) or via intervening non-canonical pairs, including sheared or Watson-Crick base pairs that facilitate the stacking. The geometry of coaxial stacking is optimized by specific step parameters at the interface, including slide (perpendicular displacement of base pairs) and shift (along-axis displacement), which deviate from canonical A-form values to maximize overlap and minimize steric clashes. For instance, simulations reveal a reduced twist angle (~28° vs. 33° in A-form), decreased shift (0.2 Å vs. 0.6 Å), and slightly increased slide ( -1.2 Å vs. -1.5 Å) at the stacking junction, promoting stable alignment. A prominent example is found in (tRNA), where the acceptor stem and T stem form a coaxial stack, contributing to the characteristic L-shaped tertiary fold by aligning their axes nearly continuously. The energetic stabilization of coaxial stacking arises primarily from van der Waals attractions between stacked bases and hydrophobic effects that exclude water from the interface, with enthalpic contributions dominating over entropic terms. Experimental nearest-neighbor parameters indicate free energy changes (ΔG°₃₇) of approximately -2.0 to -3.0 kcal/mol per interface, though total enthalpic stabilization (ΔH°) can reach -8 to -12 kcal/mol, reflecting the strength of these non-covalent forces; in some multi-interface cases, cumulative effects approach 5 kcal/mol per stack. Coaxial stacks are classified into continuous and kinked types based on the angular deviation at the junction. Continuous stacks maintain a nearly straight helical axis with minimal bending (kink angles <5°), as seen in the nicked motifs or tRNA arms, while kinked stacks introduce bends of 4° to 26°, often mediated by sequence-specific distortions that allow flexibility in larger architectures.

Base stacking in non-helical regions

In non-helical regions of nucleic acids, such as loops, bulges, and junctions, base stacking occurs through non-canonical arrangements where tandem bases from single-stranded segments interact without forming continuous helical axes. These interactions often involve , where a central intercalates between two flanking bases, or base-wedged elements (BWEs), where a nucleotide wedges into the stack at a non-adjacent position, creating zipper-like motifs that compact flexible regions. Such tandem stacking deviates from canonical helical geometry and is characterized by variations in roll (base pair opening along the major groove) and propeller twist ( inclination relative to the axis) angles, facilitating irregular overlaps and enhancing local flexibility. The stability of these non-canonical stacks primarily arises from π-π interactions between the aromatic rings of nucleobases, which provide hydrophobic and electrostatic stabilization, comparable to helical stacking but with greater context dependence. Sequence preferences favor purine-purine pairs, such as adenine-adenine or guanine-adenine, due to their larger planar surfaces and higher electron delocalization, occurring in approximately 50% of observed cases, while purine-pyrimidine stacks are less frequent and pyrimidine-pyrimidine rare. These preferences are evident in modified , which appear in about 2% of stacks but enhance stability in functional contexts. In (rRNA), non-canonical base stacking in non-helical regions exemplifies interdomain compaction, as seen in the 16S rRNA where a BIE involving adenines A1318-A978-A1319 folds the 3'-major domain, bridging helices separated by over 50 in the primary . Similarly, in 23S rRNA, stacks near modified base m²A2503 in bulge loops mediate domain-domain contacts essential for function and binding. These interactions contribute to the global fold by bridging distant secondary structure elements, such as helices and loops, thereby reducing and stabilizing the tertiary architecture without relying on alignments.

Tertiary Motifs

Loop and receptor interactions

Loop and receptor interactions represent a fundamental class of tertiary contacts in nucleic acids, particularly , where small loops dock onto complementary receptor sites to stabilize compact three-dimensional architectures. These interactions often involve tetraloops—four-nucleotide loops capping helical stems—that engage with receptor helices through , such as sheared G-A pairs between the first of the loop and an in the receptor. This motif was first structurally characterized in the P4-P6 domain of the thermophila group I , where a GAAA tetraloop binds an 11-nucleotide receptor in the J6a/6b joining , forming up to 10 bonds that mimic Watson-Crick in strength. The docking exposes the minor groove of the receptor , enabling precise alignment and contributing significantly to the overall folding stability of large molecules. GNRA tetraloops (where N is any and R is a ) are prevalent in these interactions due to their sequence-specific recognition by helical receptors, often involving A-minor motifs where adenines insert into the receptor's minor groove. In evolution studies using a group I intron system have demonstrated that receptors can be selected to recognize specific GNRA loops, revealing rules for compatibility such as complementary non-canonical pairs and loop geometry. For instance, the GAAA variant forms robust contacts in group I introns, while sequence variants like GUAA show similar binding affinities when paired with engineered receptors. Certain tetraloops, such as UUCG, exhibit exceptional intrinsic stability independent of the closing stem sequence, attributed to a cross-loop U-G and extensive stacking interactions, making them favored in natural RNAs for both autonomous folding and receptor engagement. Kissing loops constitute another modular tertiary interaction, where loops from symmetric hairpin structures pair via complementary base pairing, typically involving 4–6 nucleotides to form a transient or stable complex. These are prominent in viral genomes, such as the dimerization initiation site (DIS) of HIV-1, where two palindromic loops from stem-loop 1 () form a kissing complex with six consecutive Watson-Crick pairs, initiating genomic dimerization essential for packaging and replication. The interaction is highly specific, with mutations disrupting loop complementarity abolishing dimer formation . Similar kissing motifs occur in other retroviruses and bacteriophages, underscoring their role in RNA-RNA recognition. The modular nature of loop-receptor interactions allows interchangeability in functional RNAs, particularly ribozymes. In group I introns, the tetraloop-receptor pair in the P4-P6 domain can be swapped with compatible variants without disrupting catalysis, as shown by in vitro selections that isolated novel receptors for GNRA loops while preserving splicing activity. This plug-and-socket-like modularity facilitates RNA evolution and engineering, enabling the assembly of larger architectures from smaller, stable modules. Kissing loops similarly exhibit modularity, as demonstrated by their use in reconstructing split ribozymes where loop pairing restores function. Overall, these interactions provide versatile building blocks for nucleic acid tertiary structure, balancing specificity and stability through non-canonical pairing and geometric complementarity.

Insertion and groove motifs

Insertion and groove motifs in nucleic acid tertiary structure involve the insertion of or groups into the minor or major grooves of RNA helices, facilitating interhelical packing and stabilization. These motifs exploit the geometry of helical grooves to enable precise bonding interactions that contribute to the overall folding of molecules. The A-minor motif is a prevalent insertion motif where the base inserts its Watson-Crick face into the minor groove of a neighboring , forming bonds primarily with the 2'-OH groups of sugars in the receiving base pairs. This interaction is classified into types based on the positioning of the adenine's N3 atom and O2' relative to the groove: Type I features both the adenine's N3 and O2' within the minor groove, maximizing contacts; Type II positions the N3 inside the groove but the O2' outside, interacting with the near strand's 2'-OH; and Type 0 involves the N3 outside the far strand's 2'-OH with minimal specificity. In the large ribosomal subunit, A-minor motifs are highly abundant, with 186 adenines participating in such interactions in the 23S and 5S rRNAs of Haloarcula marismortui, underscoring their role in stabilizing core RNA architecture. The ribose zipper motif complements groove insertions by forming a chain of inter-strand hydrogen bonds between consecutive 2'-OH groups across two RNA strands, often adjacent to A-minor motifs, to enforce close packing of helices. Each "zipper tooth" typically involves two hydrogen bonds from a 2'-OH donor to a phosphate oxygen and a base edge acceptor, contributing approximately -1.0 kcal/mol to the folding free energy with additive effects across the motif. This motif was first identified in the P4-P6 domain of the Tetrahymena group I intron, where it mediates coaxial stacking of helices J6a and P5. Ribose zippers are also conserved in ribosomal RNAs, where they bridge chain segments and interact with ribosomal proteins via basic residues. In the ribosome's decoding center, A-minor motifs play a critical role in tRNA-mRNA recognition, where conserved adenines A1492 and A1493 from the 16S rRNA flip out to contact the minor groove of the codon-anticodon helix, stabilizing base pairing and ensuring fidelity. Similarly, in tRNA-mRNA interactions at the ribosomal A site, the 3'-terminal of tRNA forms a Type I A-minor motif with 23S rRNA elements, aiding precise positioning during decoding. These motifs enable RNA-RNA recognition by providing shape complementarity and hydrogen bonding specificity, while also stabilizing active sites in ribozymes and ribonucleoprotein complexes.

Pseudoknots and kissing loops

Pseudoknots represent a fundamental class of RNA tertiary structures where a single-stranded loop from one base-pairs with a complementary sequence outside the enclosing helices, resulting in intertwined helical stems that cross each other. This architecture creates a distinct from simple helices, with the H-type being the most common variant, characterized by two stems (S1 and S2) connected by two loops (L1 and L2), where L1 bridges S1 and S2 while L2 spans the minor groove of S1. More complex topologies, such as three-stemmed pseudoknots, involve additional base-pairing bridges that increase structural intricacy and functional versatility. Kissing loops constitute a specialized form of arising from direct base-pairing between the apical loops of two separate hairpins, often forming loop-loop pseudoknots that can extend beyond initial pairwise contacts to rearrange into more elaborate pseudoknot-like configurations. In these interactions, complementary in the loops—typically 2 to 4 base pairs—create a transient or stable tertiary contact, which may evolve into crossed-strand pairings involving adjacent stems, thereby mimicking the topology of H-type pseudoknots. Such extensions are facilitated by magnesium ions, which promote conformational shifts in the participating hairpins. The structural complexity of pseudoknots is often quantified by their crossing number, which denotes the number of strand interchanges between stems, with H-type pseudoknots exhibiting a crossing number of 1 and higher-order variants reaching 2 or more. Connectivity diagrams illustrate these topologies as interconnected loops and stems, highlighting how L1 typically adopts an extended conformation to pair across stems, while L2 forms compact minor-groove motifs stabilized by non-canonical base triples and ion coordination. Overall stability derives from the coaxial stacking of multiple stems, reinforced by tertiary base triples (e.g., Hoogsteen and Watson-Crick edges) and hydrogen bonding networks that exceed those in simple helices, often yielding mechanical strengths up to 30 pN in minimal kissing complexes. In biological contexts, pseudoknots play critical roles in and , such as in the human telomerase RNA pseudoknot, where conserved tertiary triples between uridine- and adenine-rich loops encircle a helical junction to maintain activity. They are prominently involved in programmed ribosomal frameshifting, as seen in viruses like beet western yellows virus (BWYV), where the pseudoknot's bent quasi-continuous and minor-groove triplex stimulate -1 frameshifting to produce fusion proteins essential for expression. Similarly, H-type pseudoknots in SARS-CoV and HIV-1 RNAs enhance frameshifting efficiency through their mechanical rigidity and loop- interactions, ensuring balanced production of viral proteins.

Stabilizing Factors

Role of metal ions

Metal ions play a crucial role in stabilizing the tertiary structures of nucleic acids, particularly , by neutralizing the negative charges on backbones and facilitating specific folding motifs. Divalent cations such as Mg²⁺ and Ca²⁺ are essential for coordinating directly with groups and nucleobases in active sites, enabling the formation of compact tertiary architectures. For instance, in the hammerhead ribozyme, two Mg²⁺ ions position near the cleavage site to stabilize the folded conformation and promote through coordination to non-bridging oxygens of the scissile . These ions reduce electrostatic repulsion between negatively charged s, allowing the to adopt its functional tertiary structure. Monovalent cations like K⁺ and Na⁺ primarily contribute to stability through electrostatic screening rather than direct coordination in active sites. In G-quadruplex structures, K⁺ ions occupy the central channel formed by stacked G-tetrads, dehydrating partially to shield phosphate repulsions and enhance folding kinetics and thermal stability. Na⁺ can similarly stabilize these quadruplexes, though with lower specificity and affinity compared to K⁺, influencing the and persistence of the structure in physiological conditions. This screening effect is vital for maintaining the folded state against the inherent electrostatic barriers in densely packed assemblies. Metal bind to nucleic acids via distinct modes that dictate their stabilizing function. Inner-sphere binding involves direct ligation of the metal to ligands, such as oxygens or nucleobase atoms, often requiring partial dehydration and occurring at high-affinity sites in catalytic cores. In contrast, outer-sphere binding is water-mediated, where the fully or partially hydrated interacts electrostatically without direct contact, commonly seen in structural stabilization. Binding can also be classified as site-specific, involving precise positioning at motifs like loops or junctions, versus diffuse, where delocalize along the backbone to broadly counter repulsion. In group II introns, a combination of these modes—such as site-specific inner-sphere Mg²⁺ coordination in the —facilitates tertiary folding and splicing, with diffuse aiding overall electrostatic balance. These interactions exemplify how metal enable the transition to stable tertiary states by modulating electrostatic forces.

Non-covalent interactions

Non-covalent interactions, including hydrogen bonding, van der Waals forces, and hydrophobic effects, play essential roles in stabilizing the tertiary structure of nucleic acids by linking distant structural elements and shielding components from the aqueous environment. These forces enable the folding of RNA and DNA into compact, functional architectures beyond secondary helical motifs, overcoming the inherent flexibility and electrostatic challenges of the phosphodiester backbone. In RNA, for instance, these interactions facilitate the formation of motifs like pseudoknots and loops that define catalytic and regulatory functions. Hydrogen bonds, particularly those involving non-Watson-Crick base pairs, are crucial for bridging remote regions in tertiary structures. Sheared G·A pairs, a common non-canonical motif, form through two hydrogen bonds between the guanine amino group and N7, as well as guanine N3 and N6, allowing parallel alignment that connects helices or loops separated by dozens of . These pairs are recurrent in and ribozymes, contributing to overall rigidity without disrupting helical continuity. Individual hydrogen bonds in such RNA base pairs typically contribute 2-5 kcal/mol to stability, with sheared G·A interactions estimated at around 4-6 kcal/mol based on quantum mechanical calculations of similar non-canonical geometries. Van der Waals interactions provide subtle but cumulative stabilization through close-range attractions between atoms in the backbone and bases, promoting the burial of non-polar surfaces. These dispersion forces, arising from transient dipoles, are particularly important in non-helical regions where atoms approach within 3-4 , enhancing packing efficiency in compact folds like the core of . In tertiary structures, van der Waals contacts often outnumber bonds, contributing approximately 0.5-1 kcal/mol per interaction and collectively mitigating exposure. Hydrophobic effects drive the exclusion of from the interior of folded nucleic acids, favoring the burial of apolar base surfaces and moieties to minimize unfavorable loss in the surrounding . This is analogous to but acts primarily on the nucleobases, which have hydrophobic faces that cluster in tertiary cores, as seen in the P4-P6 domain of the Tetrahymena ribozyme where desolvation enhances motif assembly. The energetic gain from hydrophobic burial can exceed 10 kcal/mol for multi-base clusters, underscoring its role in promoting long-range contacts essential for functional three-dimensional architectures. Electrostatic interactions, independent of metal ions, influence tertiary folding by balancing the repulsive forces among negatively charged groups along the backbone. In the absence of cations, these repulsions—stemming from the partial charges on oxygen atoms—can destabilize extended conformations, but non-ionic attractions like those from polarized bonds and induced dipoles help compact the , reducing effective inter- distances in motifs such as A-minor interactions. This mitigation is critical in low-salt conditions, where folding relies on organic forces to counteract repulsion energies estimated at approximately 1-2 kcal/mol per adjacent pair.

Experimental and Computational Methods

Experimental techniques

has been instrumental in providing high-resolution atomic models of nucleic acid tertiary structures, particularly for stable, crystalline forms such as domains. Landmark structures include the 2.4 Å resolution model of the large ribosomal subunit from Haloarcula marismortui, which revealed intricate tertiary interactions like coaxial stacking and base triples stabilizing the core. This technique excels at resolving non-helical motifs in RNAs up to several hundred , but it faces challenges with inherent molecular flexibility, often requiring stabilization by metal ions or ligands to obtain diffraction-quality crystals. Nuclear magnetic resonance (NMR) complements by elucidating tertiary structures in solution, capturing dynamic ensembles for smaller molecules typically under 50-100 . Key distance restraints from (NOE) , along with torsion angle measurements from , enable the determination of motifs like pseudoknots in transfer RNAs and domains. For instance, NMR has mapped the tertiary fold of the P4-P6 domain in the , highlighting hydrogen bonding networks and base stacking that drive folding. While powerful for studying conformational flexibility, NMR is limited by spectral overlap in larger systems and requires for signal enhancement. Cryogenic electron microscopy (cryo-EM) has revolutionized the visualization of large nucleic acid-containing complexes, achieving near-atomic resolutions for flexible assemblies previously intractable by other methods. In spliceosomes, cryo-EM structures post-2020 have reached 3.3-3.5 resolution, detailing RNA-protein interfaces and dynamic rearrangements during splicing, such as U2 and U6 snRNA tertiary contacts. Advances in detector technology and image processing have improved resolutions from ~4 in early structures to sub-3.5 routinely, enabling de novo modeling of RNA backbones in megadalton-scale ribonucleoprotein particles. This method is particularly suited for capturing heterogeneous states in native-like conditions without . Chemical probing techniques, such as selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) and dimethyl sulfate (DMS) footprinting, map tertiary contacts by assessing nucleotide reactivity in structured RNAs. SHAPE reagents like 1M7 modify flexible 2'-OH groups in unpaired or strained positions, providing single-nucleotide resolution data that infer base-pairing and long-range interactions in motifs like kissing loops. DMS selectively alkylates unpaired adenines and cytidines at the N1 and N3 positions, respectively, revealing protection patterns in tertiary folds, as demonstrated in ribosomal RNA where it highlights groove insertions and ion-binding sites. These methods are adaptable to high-throughput sequencing (e.g., SHAPE-MaP, DMS-MaPseq) for in vivo probing, offering indirect validation of tertiary models from biophysical techniques.

Prediction and modeling approaches

Traditional approaches to nucleic acid tertiary structure prediction often begin with secondary structure modeling, followed by assembly into three-dimensional folds. The MC-Fold/MC-Sym pipeline, for instance, uses sampling to generate secondary structures from sequence data based on thermodynamic parameters, then constructs tertiary models by incorporating known motifs and constraints from experimental data. Similarly, RNAstructure software employs dynamic programming for free energy minimization to predict secondary structures, which can be extended in pipelines to tertiary modeling via fragment assembly or simulation refinement. (MD) simulations complement these by exploring conformational dynamics, using atomistic force fields to relax initial models and capture tertiary interactions like base stacking and hydrogen bonding over picosecond to timescales. Recent advances in and have transformed the field, enabling end-to-end predictions from sequence alone. AlphaFold3, released in 2024, extends deep learning architectures to model RNA tertiary structures, including single chains up to thousands of nucleotides, by predicting joint atomic coordinates through diffusion-based generative networks trained on (PDB) entries. It achieves median RMSD values around 8-9 Å for many RNA targets, with some cases below 4 Å but overall showing limitations for RNA compared to proteins, while outperforming some physics-based methods in capturing long-range tertiary contacts. RoseTTAFoldNA, an adaptation of the RoseTTAFold framework from 2023, predicts structures in isolation or complex with proteins using a three-track that integrates sequence, evolutionary, and geometric features, yielding template-free models with TM-scores around 0.7 for RNA-protein interfaces. These methods leverage large-scale PDB training data, including over 200,000 RNA-containing structures, to infer tertiary motifs like pseudoknots and loops via attention mechanisms and graph neural networks. Despite progress, challenges persist in modeling long-range interactions and inherent dynamics, as RNA flexibility often leads to multiple conformations not fully resolved by static predictions. Benchmarks such as the Critical Assessment of Structure Prediction (CASP15) RNA targets from 2023 highlight these issues, where top predictors achieved combined Z-scores up to 2.5 but struggled with RNAs longer than 100 , with average RMSDs exceeding 5 for dynamic regions. From 2023 to 2025, innovations have focused on integrating tertiary contacts for more accurate designs. RhoFold+, a 2024 language model-based method, predicts RNA 3D structures with an average RMSD of about 4 on benchmarks like RNA-Puzzles by encoding sequences into geometric representations and refining via autoregressive generation. NuFold, introduced in 2025, employs end-to-end to directly output tertiary folds, achieving average RMSD values around 6-7 and accurate recovery of complex motifs like pseudoknots in case studies through hierarchical on interaction graphs. These tools emphasize complex-aware modeling, enabling de novo design of functional tertiary structures validated against experimental PDB entries.

Biological Roles

In catalysis and function

Nucleic acid tertiary structures are essential for the catalytic activity of ribozymes, where precisely folded domains create active sites that align substrates and cofactors for phosphodiester bond formation or cleavage. In group I self-splicing introns, the P4-P6 domain serves as a key tertiary scaffold, independently folding into a compact structure that organizes the catalytic core by coaxially stacking helices P4, P5, and P6, connected by a network of non-Watson-Crick base pairs and a GAAA tetraloop-receptor interaction. This architecture positions the internal guide sequence (IGS) adjacent to the guanosine binding site, enabling the first transesterification reaction by aligning the 5' splice site with the exogenous guanosine cofactor. Mutations disrupting these tertiary contacts impair substrate positioning and splicing efficiency, underscoring the domain's role in catalysis. Riboswitches exemplify how tertiary folding modulates regulatory functions through ligand-induced conformational changes. In the thiamine pyrophosphate (TPP) riboswitch, binding of the TPP ligand stabilizes a tertiary platform formed by the juxtaposition of helices P1 and P3 via base triples and a three-way junction, which propagates structural rearrangements to the downstream expression platform, thereby repressing transcription or . This folding pathway involves initial secondary structure formation followed by ligand-dependent tertiary compaction, as revealed by single-molecule FRET studies showing a transition from an open to a closed state upon TPP association. The resulting tertiary interactions sequester regulatory sequences, providing a mechanism for metabolite sensing without protein involvement. Conformational dynamics within tertiary structures enable sequential functional steps in catalytic nucleic acids. In group I self-splicing, dynamic switches between docked and undocked states of peripheral domains relative to the core facilitate the transition from the first to the second transesterification reaction; for instance, transient undocking of the P1 helix after attack allows repositioning for 3' splice site cleavage. These switches are governed by Mg²⁺-dependent tertiary contacts that stabilize intermediate conformations, ensuring in the two-step mechanism. Tertiary elements in CRISPR guide RNAs (gRNAs) contribute to the endonuclease activity of Cas9 by structuring the RNA for protein interaction and target recognition. The sgRNA folds into a compact tertiary architecture with three helical stem-loops (SL1, SL2, and SL3) that grip the Cas9 REC lobe, positioning the 20-nucleotide spacer sequence in a groove for base-pairing with target DNA while maintaining PAM-proximal contacts. This organization activates the HNH and RuvC nuclease domains for coordinated double-strand cleavage, with disruptions to SL1-SL2 tertiary packing reducing editing efficiency. In DNA, tertiary structures such as supercoiling play critical roles in biological functions by regulating access to genetic information. Negative supercoiling facilitates unwinding of , promoting processes like transcription initiation and replication fork progression, while positive supercoils generated during these activities are relieved by topoisomerases to prevent torsional stress. Nucleosome-based tertiary packaging influences through chromatin accessibility, with modifications altering higher-order folding to enable or repress transcription in eukaryotic cells.

In molecular recognition and therapeutics

The tertiary structure of nucleic acids enables precise molecular recognition by forming unique three-dimensional pockets and surfaces that interact with proteins, other nucleic acids, and small molecules. In the ribosome's A-site decoding center, the tertiary arrangement of 16S rRNA helix 44 and helix 34 creates a binding pocket that accommodates the anticodon stem-loop of , facilitating codon recognition and ensuring translational fidelity through specific hydrogen bonding and stacking interactions. This pocket is further stabilized by ribosomal proteins like S12, which recognize the RNA's tertiary fold to modulate decoding accuracy. Similarly, in RNA-protein complexes, tertiary motifs such as pseudoknots in viral RNAs bind regulatory proteins, where the 3D architecture dictates specificity beyond sequence alone. In therapeutics, nucleic acid tertiary structures underpin the design of aptamers, which fold into compact 3D conformations to achieve high-affinity binding for targeted interventions. For instance, aptamers like AS1411 form tertiary structures that selectively bind nucleolin on cancer cells, enabling tumor-specific delivery of conjugated drugs such as . These stable folds confer resistance to nucleases and enhance compared to unstructured . In siRNA therapeutics, designs incorporating tertiary-stabilizing motifs, such as base-modified overhangs that promote higher-order interactions, improve RISC loading and efficiency, as seen in optimized formulations for hypercholesterolemia treatment. Advances in mRNA vaccines from 2020 to 2025 have leveraged structured untranslated regions (UTRs) with stem-loop structures derived from viral elements to enhance stability and expression. In vaccines like BNT162b2, engineered 5' UTRs incorporate these stem-loop structures to optimize ribosomal recruitment and mRNA half-life , contributing to robust immune responses. These designs mitigate degradation by cellular RNases, with structural stabilization increasing yields by up to 10-fold in preclinical models. A key challenge in exploiting tertiary structures for therapeutics is maintaining stability , where physiological conditions like ionic fluctuations and enzymatic activity can disrupt folds. G-quadruplexes, prevalent in promoters, exemplify this: targeting s like TMPyP4 stabilize these structures to downregulate genes such as c-MYC in models, inducing . However, efficacy is limited by rapid ligand dissociation and off-target binding, with stability half-lives often under 24 hours in serum, necessitating conjugation strategies for prolonged activity. Looking ahead, AI-driven approaches to design custom tertiary scaffolds hold promise for applications. Machine learning models like those predicting RNA 3D s from sequence enable the creation of stable, multifunctional RNAs for targeted delivery, such as scaffolds encapsulating components with enhanced nuclease resistance. These tools, integrated with experimental validation, could yield next-generation vectors for treating genetic disorders by optimizing tertiary interactions for cellular uptake and payload release. tools briefly aid this by simulating energies to refine designs.

Historical Development

Early discoveries

Early investigations into the three-dimensional architecture of nucleic acids relied on X-ray fiber diffraction techniques, which provided initial glimpses of ordered structures beyond linear sequences. In the late 1940s and early 1950s, Rosalind Franklin's work at King's College London produced high-resolution diffraction patterns of DNA fibers, revealing distinct A and B forms that indicated helical conformations and suggested the potential for more complex three-dimensional arrangements in hydrated conditions. These observations, captured in her famous Photograph 51, demonstrated the helical pitch and base stacking in DNA, laying groundwork for understanding tertiary folding in nucleic acids. The 1953 double helix model proposed by James Watson and Francis Crick built upon Franklin's data, establishing the antiparallel double-stranded secondary structure of DNA as a twisted ladder stabilized by base pairing and stacking interactions. This model implied that nucleic acids could form higher-order structures, influencing subsequent RNA studies. During the 1950s and 1960s, efforts focused on sequencing and functional roles, but structural insights advanced significantly in 1974 with the first crystal structure of yeast phenylalanine transfer RNA (tRNA^Phe), determined at 3 Å resolution by Sung-Hou Kim and colleagues. This revealed the iconic L-shaped tertiary fold of tRNA, where two helical domains stack perpendicularly, connected by non-canonical base pairs and hydrogen bonds that stabilize the compact 3D architecture essential for its adaptor function in protein synthesis. Independently, Alexander Rich's group reported a similar structure, confirming the conserved tertiary motif across tRNAs. The 1980s marked a pivotal shift with the discovery of catalytic RNAs, or , which underscored the functional importance of tertiary structures. In 1982, Thomas Cech's laboratory demonstrated that the group I from thermophila pre-rRNA could self-splice without proteins, revealing RNA's enzymatic capability and the necessity of precise tertiary folding for . This finding, detailed in a seminal Cell paper, showed autocyclization and excision driven by the intron's folded core. Shortly after, in 1983, Sidney Altman's group established that the RNA subunit of RNase P performs the catalytic cleavage, further evidencing RNA's active role in tertiary-configured complexes. These discoveries prompted the identification of recurring RNA tertiary motifs, such as coaxial helical stacking and non-Watson-Crick base triples, initially modeled from tRNA and expanded in ribozyme analyses during the mid-1980s. A key milestone in understanding tertiary folding came in 1986, when studies on group I introns revealed mechanisms of structure assembly. Research on the mitochondrial large rRNA demonstrated protein-assisted splicing in ribonucleoprotein particles, highlighting how maturase proteins facilitate the formation of the active tertiary core by stabilizing intermediate folds. This work illustrated the magnesium-dependent compaction of distant secondary elements into a functional 3D scaffold, advancing models of intron self-splicing and folding pathways.

Modern milestones

The determination of atomic-resolution crystal structures of ribosomal subunits in 2000 represented a pivotal breakthrough in visualizing nucleic acid tertiary architecture within large ribonucleoprotein complexes. Thomas A. Steitz and colleagues resolved the 50S large subunit from Haloarcula marismortui at 2.4 Å, unveiling a compact RNA core with extensive tertiary contacts, including pseudoknots and coaxial helices that stabilize the peptidyl transferase center. Concurrently, Venkatraman Ramakrishnan's team reported the 30S small subunit structure at 3 Å resolution, illuminating the decoding site's tertiary folds and intersubunit bridges essential for translation fidelity. These structures, which earned the 2009 Nobel Prize in Chemistry, shifted paradigms by demonstrating RNA's catalytic role and the prevalence of non-canonical tertiary motifs in functional complexes. In 2001, classification of the A-minor motif further refined understanding of recurrent RNA tertiary interactions observed in ribosome structures. Peter Nissen, Jaime A. Ippolito, and colleagues identified this motif as the insertion of adenine's minor-groove edges into adjacent helices, forming hydrogen bonds that mediate packing and stability across diverse contexts, such as group I introns and the . This motif, now recognized as one of the most common tertiary elements, accounted for over 10% of interhelical contacts in known RNA structures at the time, providing a framework for annotating similar interactions in emerging datasets. The 2010s brought the cryo-EM resolution revolution, enabling near-atomic visualization of large, flexible RNAs previously intractable to . Advances in direct electron detectors and phase plates allowed resolutions below 4 Å for ribonucleoprotein assemblies, such as the human and bacterial ribosomes in functional states, revealing dynamic tertiary rearrangements during catalysis. For instance, cryo-EM structures of the at 3.3–3.5 Å highlighted RNA-mediated tertiary scaffolds that coordinate protein factors. This era exponentially increased the number of solved RNA tertiary structures, from dozens to hundreds, emphasizing conformational heterogeneity . A landmark computational analysis of (G4) motifs identified approximately 376,000 potential G4-forming sequences in the , with enrichment in promoters and telomeres. Julian L. Huppert and demonstrated that these non-canonical tertiary folds, involving stacked G-tetrads stabilized by monovalent cations, are evolutionarily conserved and associated with regulatory hotspots, influencing and replication. This work validated as widespread tertiary elements, spurring experimental validations and therapeutic targeting. From 2020 to 2025, transformed tertiary structure prediction, with models achieving de novo atomic-level accuracy for sequences up to 200 . In 2022, Robert Pearce and Yang Zhang introduced DeepFoldRNA, coupling self-attention neural networks with physics-based simulations to predict tertiary folds without templates, outperforming traditional methods on benchmarks like the RNA-Puzzles dataset by reducing RMSD errors by up to 30%. Building on this, the 2022 Critical Assessment of Structure Prediction (CASP15) RNA targets showcased AI's potential, though challenges persisted for longer RNAs. In 2024, CASP16 further advanced RNA predictions, with top methods achieving median GDT-TS scores above 60 for tertiary structures. Additionally, AlphaFold3 extended multimodal predictions to include nucleic acids, enabling accurate modeling of -protein complexes. Recent advances in chemical probing illuminated RNA dynamics, with 2024 developments in DMS (dimethyl sulfate) mapping providing quantitative metrics for 3D structural features. Yu-Ming Jhang and colleagues established relationships between DMS reactivity and RNA tertiary elements, distinguishing base-pairing states and applied to diverse motifs including riboswitches, revealing conformational fluctuations. This enhanced single-molecule resolution, integrating with cryo-EM for hybrid dynamic models. The accelerated insights into mRNA tertiary structures for therapeutics, as structural optimization of lipid nanoparticle-encapsulated mRNAs improved vaccine efficacy. Studies of Pfizer-BioNTech and vaccines revealed that 5′ and 3′ folds, including stem-loops, shield against degradation while promoting translation, with modifications stabilizing tertiary motifs to evade innate immunity. Cryo-TEM analyses confirmed compact mRNA conformations within nanoparticles, informing iterative designs that boosted protein expression by 2–5 fold. These milestones have vastly expanded the catalog of solved tertiary structures, from isolated motifs to genome-scale distributions, yet capturing transient dynamics remains a key gap. While static snapshots abound—over 5,000 RNA-containing entries in the PDB by 2025—methods like DMS and cryo-EM struggle with heterogeneous ensembles, limiting full atomic models of functional trajectories.

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