In this Opinion, we argue that the triple helix is a protein structure of fundamental importance in building the extracellular matrix, which enabled animal multicellularity and tissue evolution. The extracellular matrix ECM played an essential role during the transition from unicellular organisms to multicellular animals metazoans. The ECM comprises a basement membrane BM that underlies epithelia cells, and an interstitial matrix IM that is positioned between cells in the intercellular spaces and undergoes continuous controlled remodeling Hynes, ; Bonnans et al.
Yet, a major gap in cell biology is to understand how cells generate and interact with the ECM Sherwood, ; Jayadev and Sherwood, Among these collagens, type IV is the evolutionarily most ancient, based on recent studies of non-bilaterian animals sponges, ctenophores, placozoans and cnidarians and unicellular groups Fidler et al.
Collagen IV is the evolutionarily most ancient of the vertebrate collagen superfamily. A hallmark feature of all collagens is the triple helix, which is characterized by three intertwined polypeptide chains. A collagen IV-like gene probably first appeared in the last common ancestor LCA to filastereans, choanoflagellates and animals.
The phylogenetic distribution suggests that collagen IV played a critical role in the transition of unicellular organisms to multicellular animals. Collagens are the most abundant protein in the human body Kadler et al.
They occur as diverse supramolecular assemblies, ranging from networks to fibrils, and broadly function in structural, mechanical and organizational roles that define tissue architecture and influence cellular behavior Shoulders and Raines, ; Ricard-Blum, ; Ricard-Blum and Ruggiero, Defects in collagens underlie the cause of almost 40 human genetic diseases, affecting numerous organs and tissues in millions of people worldwide summarized in Table 1.
Disease pathogenesis typically involves genetic alterations of the triple helix, a unique structure that is a hallmark feature common to all collagens. The triple helix bestows exceptional mechanical resistance to tensile forces and a capacity to bind a plethora of macromolecules. Yet, there is a gap in our current knowledge in understanding the mechanisms of how a triple helix encodes and utilizes information in building supramolecular assemblies on the outside of cells.
Here, we present collagen IV, the most ancient of the collagen superfamily, and argue that it is ideally suited to serve as an archetype for investigating and describing core functions of a triple helix. The chemical structure of the triple helix was determined through the seminal work of structural biologists and chemists over the last century see Box 1 in supplementary material.
Its unique structure bestows upon collagens an exceptional mechanical resistance to tensile forces and a plethora of organizing information for building an ECM Fig. The triple helix presents all residues, except glycine Gly , on its surface, which is the most economical and robust way to encode binding motifs of any protein structure. Moreover, the triple helix exhibits extensive post-translational modifications PTMs , such as hydroxylation, glycosylation and phosphorylation, adding — in tandem — a secondary layer of information in addition to its amino acid aa code Yamauchi and Shiiba, These PTMs confer even more diversity with tissue-specific and disease-specific variations, even amongst identical types of collagen Pokidysheva et al.
Furthermore, additional collagen modifications are mediated by specific extracellular enzymes, such as peroxidasin and lysyl oxidases-like proteins LOXLs for crosslinking, and Goodpasture antigen-binding protein COL4A3BP, hereafter referred to as GPBP and other extracellular kinases for phosphorylation Bhave et al.
Non-enzymatic modifications, such as glycation, oxidation or chlorination, add even more complexity Brown et al. Together, these modifications may serve as regulatory mechanisms on the outside of cells that may instruct cell behavior and influence tissue architecture and stability Yalak and Olsen, ; Pedchenko et al.
Building a collagen triple helix and encoding information. A The building blocks polypeptide chains of the triple helix are left-handed polyproline type-II helices, i. Replacement of every third proline Pro residue with a glycine Gly residue results in increased flexibility. B Three left-handed superhelices wind together and pack tightly owing to these Gly residues, thereby forming a right-handed triple helix.
Once associated, the non-extensibility of the structure is restored, and the combination of three chains results in three binding modes one, two or three chains with three levels of variation to these modes, i. Together, these specify the numerous possible binding motifs that are positioned along the length of the triple helix of any of the 28 collagen types to bind various macromolecules. To fully appreciate the capacity and versatility of the triple helix, one should consider the building blocks polypeptide chains of this structure.
A unique feature of this structure is that it is non-extensible Okuyama et al. Hydra Holstein et al. To allow for the formation of a triple helix from this simple rope, every third proline residue of the PPII helix is replaced with a glycine residue — the smallest aa residue as it lacks a side chain.
This endows the structure with flexibility and sufficient space to tightly pack three chains Fig. Moreover, as these three chains wind together forming a triple helix, each individual left-handed chain adopts a right-handed superhelix.
The unique role of the glycine residues in the packing of the collagen triple helix explains the adverse effects of mutations at these positions, as any other residue in place of glycine will distort its tight packing Bella et al. Three main changes are achieved upon the transition from a single polyproline helix to a triple helix: i higher bending rigidity, ii a less accessible chain backbone that is, thus, less prone to proteolysis and, iii the ability to essentially accept any aa in place of the proline residues at position X and Y without any significant destabilization to the triple helix structure Fig.
Moreover, proline and other aa within the triple helix remain accessible to solvent and, thus, their numerous known PTMs are possible without disturbing the native helix structure. For example, probably the most important PTM is the 4 R -hydroxylation of proline residues position Y, because it substantially increases the thermal stability of the collagen triple helix Sakakibara et al. Thus, the triple helix, in contrast to a single PPII helix, confers additional capacity to specify binding partners.
The three binding modes operate by utilizing one, two or all three distinct chains of the triple helix in different combinations to directly bind a molecule Fig.
The three levels of variation to these modes add additional diversity to binding specificity on top of involving either one, two or three chains. The second level confers even more diversity because of the proclivity of collagens to become post-translationally modified and change their structure Fig.
The third level utilizes chain staggering that occurs during triple helix assembly between either two or three chains and enables new combinations of chains that can give rise to additional binding motifs Fig. In addition to these binding specificities, the triple helix also possesses other structural features that underlie its biological function.
It is non-stretchable along its longitudinal axis, providing great tensile strength to withstand all physiological mechanical loads and stresses in our body. Furthermore, the triple helix exhibits variable lengths among collagen suprastructures, such as networks, fibrils and filaments Ricard-Blum, For example, the triple helix length of fibrillar collagens results from multiple duplications of exons that are either 54 or 45 base pairs in length and encode Gly-X-Y Yamada et al.
Moreover, the terminal and internal incorporation of non-triple helical sequences e. Collectively, the binding motifs see above together with these features of a collagen triple helix underlie the diversity of biological activities of the diverse supramolecular assemblies of collagens. The last common ancestor to animals, the Urmetazoan, almost certainly reproduced by gametogenesis, underwent gastrulation during early development, had the ability for cells to differentiate both during development and as stem cells, and comprised an epithelial layer of cells forming the body of the animal — features that are still fundamental to extant animals Richter and King, ; King and Rokas, Importantly, these cellular activities ultimately required the invention of an ECM to provide a substrate for attachment and signaling cues to regulate cell behavior and function in tissue genesis and homeostasis Abedin and King, The appearance of a specialized form of ECM, the BM, coincided with the transition to multicellularity.
The BM functions in several cellular activities, including migration, adhesion, delineation of apical—basal polarity and modulation of differentiation during development Petersen et al. Importantly, understanding the makeup of BMs between different animals sheds light on the functions of proteins in the evolution of animal multicellularity and tissues.
BMs are composed of numerous proteins, vary between animals. The BM of bilaterian animals e. Potentially, there are many more components of the BM Chew and Lennon, Among these, collagen IV is a major component that is conserved among animal phyla Fidler et al.
In a recent study, we described collagen IV at the origins of animal multicellularity and in tissue evolution, as revealed by close examination of sponges, ctenophores and other non-bilaterally symmetrical animals Fidler et al.
Ctenophores and sponges have been established as the two most likely candidates to be the sister-groups to the rest of animals, based on phylogenetic analyses of genomic and transcriptomic data and cell-type evolution Ryan et al. However, the demosponges, a sponge class, lack both laminin and classic collagen IV but do contain spongins, which are short collagen IV variants Exposito et al. The order in which these variants and collagen IV first appeared is still unknown.
Despite containing fewer BM proteins than bilaterians, many sponges and ctenophores form classic BMs Fidler et al. Importantly, comparison of BM components of animals with those of unicellular lineages is key to determining their importance during the transition to multicellularity. Within choanoflagellates, the closest relatives of animals, no complete ECM proteins exist; yet, domains that are characteristic of laminins and short collagenous Gly-X-Y repeats are present King et al.
Interestingly, a recent study reported the discovery of a collagen IV-like gene in the filasterean, Ministeria vibrans , a unicellular lineage that diverged prior to choanoflagellates and animals Grau-Bove et al. This finding indicates that collagen IV has a premetazoan ancestry and a function for single cells. Collectively, these findings suggest that collagen IV played a role in the transition from unicellular organisms to multicellular animals Grau-Bove et al.
Therefore, we consider collagen IV as an archetype of collagens to describe the fundamental features of a triple helix that underlie biological functions. These scaffolds provide tensile strength, connect adjacent cells and organize supramolecular protein assemblies that are able to influence cell behavior Wang et al.
Once secreted into the extracellular space, protomers adjoin via their NC1 domains and the N-terminal 7S domains Fig. Upon association mediated through NC1- and 7S-domains, the collagen IV networks are reinforced through covalent crosslinks at both the NC1- and 7S-domain interfaces.
The triple helix of collagen IV scaffolds encodes information for the tethering of macromolecules. On the outside of cells, the NC1- and 7S-domains direct the assembly of protomers into network structures of higher order crosslinked by sulfilimine and lysyl oxidase-like protein 2 LOXL2. B Once assembled, collagen IV networks function as smart scaffolds, bestowing BMs that underlie and surround cells of several capabilities.
Vast amounts of structural information is encoded in motifs located at specific sites along the surface of the triple helix to tether macromolecules. The mode of variation is based on 20 variable aa residues on a single chain, or the combination of one, two or three chains, post-translational modifications PTMs and chain stagger see Fig.
The tethering at specific sites spatially organizes molecules along the triple helix, resulting in a populated scaffold within the BM that provides tensile strength to tissues, and influences cell behavior, adhesion and migration during tissue development and regeneration.
Partly modified, with permission Protein Science from Brown et al. Via the triple helix, scaffolds tether different extracellular molecules, i. The information for the tethering of these molecules is encoded at sites within the triple helix, and depends on the 20 variable aa residues within a single chain, or a combination of one, two or three chains, chain stagger and post-translational modifications see Fig.
The tethering of these molecules at specific sites along the triple helix spatially organizes binding partners Fig. The distribution of binding partners within a BM is not a static arrangement, and can be dynamically regulated throughout early development and beyond Inman et al. The resulting scaffold, populated with macromolecules bound to the triple helix, provides tensile strength to tissues, attaches to cells through cell-surface receptors and influences cell behavior in tissue development, function and regeneration Eble et al.
The biological importance of the triple helix is displayed in several ways. It is an ancient structure that is conserved between animals and is expressed ubiquitously in their ECMs. There are almost 40 diseases wherein mutations of glycine residues affect multiple tissues and organs in millions of people Fig. Glycine residues are crucial for the structural integrity of the triple helix see Figs 2 and 4 A ; therefore, mutated collagen molecules can assemble into faulty fibrils, networks, and other assemblies can cause tissue dysfunction.
Mutations in the triple helix can result in genetic diseases. A A single missense mutation of a glycine to another residue results in disruption of the triple helix's function.
B Glycine mutations are responsible for a number of genetic diseases involving the kidney, teeth, muscle, joints, cartilage, brain, skin, vasculature, bone, inner ear and eye in humans see Table 1.
B Diseases and disorders due to replacement of glycine residue s in the triple helix of collagens. The type of collagen is indicated in bold Roman numerals. As examples for such glycine mutations, osteogenesis imperfecta OI; also known as brittle bone disease , and Alport syndrome are two collagen-dependent genetic disorders that are well-studied Fig.
For OI, over mutations in collagen I have been described Marini et al. However, such mutations, depending on the nature of substitution as well as its location, lead to different degrees of post-translational modifications and structural destabilization of the triple helix. For example, substitutions in the first residues of collagen I are non-lethal, whereas there are two regions helix positions — and — , in which substitutions can cause lethality because they align with main ligand-binding sites for integrins, matrix metalloproteinases, fibronectin and cartilage oligomeric matrix protein Marini et al.
In Alport syndrome, the collagen IV scaffold is mutated, which leads to progressive organ failure in kidney, ear and eye Williamson, ; Hudson, ; Hudson et al. In summary, the many collagen diseases that involve glycine mutations directly demonstrate the essentiality of the triple helix for tissue architecture and function. Its essentiality is further supported experimentally by collagen knockout studies in mice Table 1. The triple helix is unique among all other protein structures — globular or fibrous — in its capacity to encode vast amounts of information that is available on its surface for utilization on the outside of cells Fig.
The triple helix, arranged in various patterns forming diverse supramolecular scaffolds, tethers and spatially organizes macromolecules, thus providing tensile strength to tissues and influencing cell behavior.
This unique structure of the triple helix with its encoded degree of information evokes an analogy to the DNA double helix Fig. The fundamental importance of a triple helix in enabling animal multicellularity and tissue evolution. A The unique structure and vast encoding properties of the collagen triple helix outside the cell left evokes an analogy to the DNA double helix inside the cell right.
B The triple helix protein structure was present in unicellular organisms and was co-opted in the form of collagen IV, enabling the transition to multicellular animals.
The triple helix was also adapted as a key feature of all members of the diverse collagen superfamily in the ECM, enabling tissue evolution. LCA, last common ancestor. The biological importance of the triple helix is also evident from the almost 40 genetic diseases and its ubiquitous presence in animals. The temperature dependence of the imino proton resonances can provide insight into the stabilities of the base pairings within the triple helix.
As the temperature is increased, the exchange rate of the imino protons with those of the solvent increases and the resonances broaden and disappear. The exchange rates of the imino protons are related to the frequency with which the base pairs open.
This frequency increases as the melting point of the RNA is approached. Peaks labeled a, b and c are the amino protons of residues C24, C26 and C29 respectively. The RNA triple helix appears to be quite stable as judged by the temperature dependence of the imino resonances. First to disappear as the temperature is increased are the relatively broad resonances with chemical shifts between This imino proton is hydrogen bonded as part of the stable tetraloop structure The remaining imino and amino resonances are associated with the hydrogen bonded protons of the Watson-Crick and Hoogsteen pairs.
As the temperature is increased, the resolved guanosine imino protons at the ends of the Watson-Crick part of the triplex G1 and G6 are less protected from solvent exchange than G3 at the center of the triplex. Of particular interest, the amino resonances of the protonated cytosines are clearly observed, even at this relatively high temperature.
This phenomenon is completely reversible upon cooling the sample. The temperature dependence of the exchangeable proton resonances is most consistent with denaturation in a single transition, from triplex to a single-stranded structure, rather than a two-step transition, proceeding from triplex to stem-loop to single strand.
Analysis of UV absorption changes upon thermal denaturation. UV absorption spectroscopy as a function of temperature was used to monitor the unfolding of the RNA triplex in a solution of 25 mM potassium phosphate, pH 4. In this respect, the NMR and UV data are in agreement, with each experimental method indicating essentially the same melting point for the RNA triplex.
The data obtained using each method NMR and UV absorption is most consistent with the melting transition occurring in a single step, without a stem-loop intermediate. As expected for a 30 nt RNA, the problems encountered in interpreting the rather complex NMR spectra are formidable and this does limit the resolution of the structural model we obtain.
However, the substantial amount of data that we have been able to interpret provides significant information regarding the triplex structure. In particular, the hydrogen bonds identified by slow exchanging imino and amino protons and ribose conformations, supported by our NMR data, provide significant constraints upon the possible structure of the triplex.
We therefore constructed a model of the RNA triplex that is consistent with 99 interproton distance restraints derived from our assigned NMR data, corresponding to five restraints per nucleotide for the 21 nt triplex model. Clearly, this model does not represent a finished high resolution structure. However, this preliminary model is useful in identifying some factors that may contribute to the stability of the RNA triplex.
An initial model was constructed as follows. Nucleotides 1—7 and 12—18 were placed exactly as they would be in an ideal A-form double helix This is entirely consistent with our observed NOEs and hydrogen bonds for these nucleotides. Nucleotides CU30 were placed along the major groove of the double helical stem so that they spanned from G1 to A7, also consistent with our observed NOEs. Loop nucleotides were not included in the model.
The initial model had a relatively high value for the X-PLOR energy function, primarily due to van der Waal's violations involving close contacts of the backbones of the purine strand and the pyrimidine third strand.
The near co-planarity of the bases is consistent with fiber diffraction data on triple helices 31 , 32 and was included in our model building to compensate for the relatively sparse NOE information.
Interatomic distances between the atoms of nt 1—7 and 12—18 were restricted to be within 1. The 1. The target function that was minimized during the simulated annealing process contained the following: i quadratic harmonic potential terms for covalent geometry, including bonds, angles, planes and chirality; ii square-well potentials for interatomic distance constraints and torsion angle restraints.
The simulated annealing procedure produced a model of the structure that has chemically reasonable values for bond lengths and angles, no significant van der Waal's violations, a low value of the X-PLOR energy function and is consistent with our NMR data. Upon close inspection of the triple helix model, it can be seen that the ribose phosphate backbones of the purine strand strand 1 and the Hoogsteen strand strand 3 are in rather close proximity.
The existence of this hydrogen bond has also been suggested in a previous model building study done in the absence of NMR-derived ribose conformation and hydrogen bonding restraints 6. Our studies demonstrate that the 30 nt RNA with the sequence shown in Figure 1 forms an intramolecular antiparallel triple helix, with the ribose groups on all three strands most likely in the C3 endo conformation.
In addition to the Watson-Crick and Hoogsteen hydrogen bonding between the bases, our structural modeling provides evidence for a possible hydrogen bond between the 2 -hydroxyl proton and a phosphate oxygen on the backbone of the purine strand. A DNA triplex lacking a 2 -hydroxyl group could not form this last hydrogen bond.
This could, at least partially, account for the higher stability reported for triplexes that contain RNA rather than DNA as the Hoogsteen paired third strand 4—6. Since the DNA third strand cannot form this hydrogen bond, it does not have as much to gain in terms of stability by having its riboses adopt the C3 endo conformation.
This hydrogen bond also cannot occur in DNA and also requires that the riboses be in the C3 endo conformation. Base stacking probably makes a substantial contribution to stabilizing the third strand in triple helices and is of course a common factor in both DNA and RNA.
Hydrogen bonding interactions between the 2 -hydroxyl proton of the third strand and phosphate oxygen of the purine strand are not possible due to the methylation at the O2 position.
The loss of possible hydrogen bonding interactions is believed to be compensated for by an increase in favorable Van der Waals interactions with the methyl groups 34 , Recently, preliminary NMR and stability studies were reported for an RNA intramolecular triplex with a substantially different sequence from the RNA used in our study 16 , A comparison of the results for the two molecules gives some additional insight into the factors that are important for stabilizing the RNA triplexes.
The RNA investigated by Liquier et al. Liquier et al. Liquier and co-workers 17 used four uridines for their second connecting loop, compared with our study, where a loop of 5 nt UCUCU was used. Despite these difference in the sequences, the melting point reported by Liquier et al. The similar stabilities observed in each study are significant in that they indicate that the stable intramolecular RNA triplexes are not artifacts induced by the choice of loop or stem sequences.
The similarity of the melting points found for the different RNA triplexes suggests that the loop sequences may not be particularly important in governing intramolecular triplex stability. This is somewhat surprising, when one considers that there is a very large increase of 8. The choice of loop sequence has also been shown to influence the melting points of DNA triplexes DNA triplex structure can be constructed by molecular modeling techniques by using coordinates that correctly take into account the sugar conformation of T,C -motif triple helices [ 39 ].
This structure is closer to a B-form DNA as reported by NMR studies [ 40 , 41 ] as compared to the structure proposed [ 42 ], based on fiber X-ray diffraction. Using molecular modeling, one can demonstrate the possibility of forming a parallel triple helix in which the single strand interacts with the intact duplex in the minor groove, via novel base interactions [ 44 ].
JUMNA uses a mixture of helical and internal coordinates valence and dihedral angles to describe nucleic acid flexibility. The helical parameters position each 3'monophosphate nucleotide with respect to a fixed-axis system. Junctions between successive nucleotides are maintained with quadratic restraints on the O5'-C5' distances.
In addition to a reduced number of variables with respect to Cartesian coordinate programs, the choice of physically meaningful variables allows large, concerted conformational moves during minimization, together with an efficient control of the structure and easy introduction of constraints or restraints. Available tools include both adiabatic mapping and combinatorial searches with respect to chosen structural parameters. The slope S, the plateau value at long distance D, and the initial value D 0 of the function are adjustable, with default values of 0.
First, the base triplets are restrained to be coplanar to avoid any possible interbase triplet interactions. Such interactions easily form during the construction of stretched helices but cannot play a role in recognition or strand exchange, since these processes are independent of the overall sequence. It has been checked out that the optimized structure of the minor-groove triplex is independent of these restraints.
For this reason preliminary studies have been limited to sequences with trinucleotide repeat. Specific restraints or constraints are needed for triplex construction and manipulation. The trinucleotide symmetry constraint implies the equivalence of the variables describing each successive group of three nucleotides.
Stretching dsDNA, so that the twist decreases and the minor groove opens have been previously achieved by restraining the distance between the terminal O3' atoms of the trinucleotide symmetry unit. This restraint has been slightly modified because the O3'-O3' distance can be altered by a lateral displacement of the backbones during strand exchange.
In a recent work, only the component of the O3'-O3' vector parallel to the helix axis was restrained. The restraints on the groove width, calibrated with the help of numerical Poisson-Boltzmann electrostatic calculations, were used to avoid groove narrowing due to the lack of explicit solvent molecules [ 47 ]. Base pair switching is studied by base rotation, using the approach defined by Bernet et al [ 48 ].
Biological applications of TFOs are compromised by fundamental biophysical considerations, as well as limitations imposed by physiological conditions. Triplex formation involves the approach and binding of a negatively charged third strand to a double-negatively charged duplex. Furthermore, triplex formation involves conformational changes on the part of the third strand, and some distortion of the underlying duplex [ 40 , 49 - 52 ].
This is necessary for the second Hoogsteen hydrogen bond, although the resultant positive charge apparently makes the more important contribution to triplex stability [ 53 ]. Pyrimidine motif triplexes containing adjacent cytosines are often less stable than those with isolated cytosines. Traditionally this has been ascribed to charge-charge repulsion effects [ 54 ], although a recent study suggests incomplete protonation of adjacent cytosines may be the critical factor [ 55 ].
All these factors impose kinetic barriers on triplex formation and reduce the stability of triplexes once formed most triplexes, even under optimal conditions in vitro , are less stable than the underlying duplex [ 57 ].
The first and foremost problem encountered in triple helical antigene strategy is the instability of the triplex formed by the TFOs under physiological conditions which consequently limits the utilization of this very fascinating strategy meant for gene correction to variable extent.
Hence various approaches and strategies have been proposed to confer stability to the triple helical structure formed. Oligonucleotide directed triple helices could be stabilized by using nucleic acid ligands that selectively stabilize triple helices. Several other intercalators [ 61 ] as well as various DNA minor groove ligands [ 62 - 64 ] have also been shown to bind to DNA triple helices.
For example triple helices can be stabilized by chemical modification of oligonucleotides such as, psoralene attached to oligonucleotides has been shown to enhance their biological activity following UV irradiation [ 65 , 66 ]. Because no structural data are available on triple helix-ligand complexes, not much is known about the interactions that direct specific intercalation into triple helices.
Pyrimidine-parallel morpholino oligonucleotides were found to be able to form a triplex with duplex target. As expected, this motif required a low pH for triplex formation, as required by the pyrimidine-parallel motif phosphodiester TFO. It may be possible to overcome this pH dependence with such substitutions as 5-methylcytosine for the cytosines in the TFO [ 70 ]. An alternative approach by which triple helices can be stabilized is via chemical modifications of oligonucleotides, such as covalent attachment of an acridine molecule [ 71 ].
It has been shown that acridine substitution strongly increases the inhibition of restrictin enzyme cleavage and also it does not impair sequence specificity for triplex formation [ 71 ].
The formation of intermolecular DNA triple helices offers the possibility of designing compounds with extensive sequence recognition properties, which may be useful as antigene agents or tools in molecular biology [ 72 ]. During the past decade, a new approach using DNA analogues, as therapeutic agents, is emerging in medicinal chemistry.
It is affected through sequence-specific binding of complementary oligonucleotides to either DNA duplex via triplex formation to inhibit production of mRNA or interfere in the translation of the latter to proteins.
Since oligonucleotides do not enter cells easily and are amenable to destruction by cellular nucleases, a variety of chemically modified analogues of oligonucleotides are being designed, synthesized and evaluated for development as therapeutic agents.
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