Programmable Assembly of Multistranded Helices in Water

Programmable Assembly of Multistranded Helices in Water | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Programmable Assembly of Multistranded Helices in Water Fabien Cougnon, Dimitri Delcourt, Reguram Arumugaperumal, Prachi Verma, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7441733/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Sequence-specific conformational changes underpin essential biological processes, from information storage to energy transduction, but are difficult to replicate in synthetic systems. Here, we present a simple approach to encode in the primary sequence of molecular strands all the information required to govern both the formation and dynamic behavior of multistranded helices. We demonstrate that the sequence of oligo( m -phenylene ethynylene) strands composed of hydrophobic phenylene and charged pyridinium residues reliably direct the formation of either static structures (e.g., a double helix) or dynamic assemblies (e.g., double and triple helices in exchange). In the latter case, transitions between different helical states can be controlled by concentration, temperature, or by the presence of anionic molecules. This minimal yet versatile design strategy lays the groundwork for the construction of adaptive supramolecular systems with programmable structure and function. Physical sciences/Chemistry/Supramolecular chemistry/Self-assembly Physical sciences/Chemistry/Organic chemistry/Structure elucidation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The primary sequence of proteins and nucleic acids defines a landscape of energetically accessible conformational states, enabling these biomolecules to undergo controlled structural transitions in response to external cues. 1 The primary sequence of DNA, for example, determines its ability to adopt a variety of higher-order structures beyond the canonical right-handed B-form, including left-handed double helices (Z-form), triple helices (H-form), and G-quadruplexes. 2 , 3 Transitions between these different states play essential roles in key cellular processes like gene expression, replication, and repair. Significant efforts have been devoted to developing synthetic analogs that mimic the structural and functional complexity of biomolecules. Synthetic multistranded helices have emerged as promising biomimetic platforms for applications such as molecular recognition, 4 – 10 switching, 11 – 17 and self-replication. 18 , 19 Their assembly typically relies on non-covalent interactions, including hydrogen bonding, 20 , 21 π–π stacking, 22 – 29 and metal coordination. 13 , 30 , 31 Despite the wide range of strategies available to control helix formation, general methods for encoding both structural organization and dynamic responsiveness directly into the primary sequence of synthetic strands have yet to be established. In a landmark study, the Flood group demonstrated sequence control over single- to double-helix transitions in aryl-triazole foldamers. 26 However, extending this approach beyond that specific system remains an open challenge. Developing broadly applicable strategies that couple sequence information to both structure and dynamics would unlock a new generation of synthetic systems with biomolecule-like functions. Here we present an approach for encoding, within the primary sequence of synthetic strands, all the information necessary to control both the assembly and dynamic behavior of multistranded helices. Figure 1 a shows the general structure of a bolaamphiphilic strand, consisting of a central hydrophobic core flanked by two permanently charged, polar termini. Let h and p denote the lengths of the hydrophobic and polar segments, respectively. In aqueous environments, such bolaamphiphilic sequences promote the self-assembly of n- stranded (where n = 1, 2, 3, …) helices, which minimize exposure of the hydrophobic surface to water by positioning the polar segments at the top and bottom of the hydrophobic core. However, these helices form only if adjacent charged segments do not stack directly above one another, as it would generate destabilizing electrostatic repulsion. Electrostatic repulsion may arise between charged components situated on the same face of the helix, or between those located on opposite faces (Fig. 1 b). Repulsion between charged segments located on the same face (e.g., the upper rim) can be avoided if the combined length of these charged segments ( n·p ) does not exceed the length of one full helical turn. Defining l as the length of strand required to complete one helical turn, this condition can be expressed as: n·p ≤ l (1) By symmetry, this same constraint applies to the charged segments located at the lower rim of the helix. Repulsion between charged segments located on opposite faces of the helix can be avoided if they are separated by a full hydrophobic layer; that is, if the combined length of hydrophobic components located at the center of the structure ( n·h ) is greater or equal to l : n·h ≥ l (2) Combining inequalities (1) and (2) yields a single inequality that defines the permissible range for the number of strands n : l/h ≤ n ≤ l/p (3) This inequality establishes a direct sequence-to-structure relationship: the primary sequence of the strand (characterized by parameters h , p , and l ) sets the geometric window within which n -stranded helices are stable. When n falls within this window, helix formation is thermodynamically favored, as it reduces hydrophobic exposure without introducing repulsive interactions. Outside this range, helix formation is disfavored due to unavoidable electrostatic destabilization. Inequality (3) can thus be used to parametrize strands capable of forming specific n -stranded helices. Results and discussion In this article, we sought to experimentally test the predictions of the model described above. Oligo( m -phenylene ethynylene)s were selected as a suitable scaffold, because their conformational behavior is well documented. 32 – 37 Moore demonstrated their strong tendency to fold into single helices several decades ago. 38 – 40 The folding process is driven by solvophobic effects and can be promoted by polar solvents such as water. 41 More recently, Berryman showed that oligo( m -phenylene ethynylene)s can also form higher-order helices. 42 – 44 Building on these foundations, we reengineered the oligo( m -phenylene ethynylene) scaffold by introducing hydrophobic and polar segments of variable lengths, and assessed whether the resulting sequences predictably yielded specific types of multistranded helices. Controlling the assembly of a single type of multistranded helix (static behavior). We first designed strand 1 (Fig. 2 a), composed of h = 3 hydrophobic phenylene residues and p = 3 polar pyridinium residues. Since oligo( m -phenylene ethynylene) helices require l = 6 aromatic residues per turn, 38 – 44 the lower and upper bounds of inequality (3) are l / h = 2 and l / p = 2, respectively. Because n = 2 is the only integer that satisfies this condition, strand 1 is predicted to exclusively assemble into a double helix. This situation is illustrated schematically in the free energy diagram shown in Fig. 2 b, where the double helix represents the only thermodynamically stable state (DG° < 0). The equilibria involved in the assembly of the double helix are shown in Fig. 2 c. In agreement with our predictions, DFT calculations performed at a BP86-D4-COSMO(water)/def2-TZVP level of theory showed that the formation of a double helix allows the outer pyridiniums to effectively shield the hydrophobic phenylenes from water. Single helix formation is disfavored, as it would necessarily bring charged pyridiniums in close contact, resulting in destabilizing electrostatic repulsion. The assembly of higher-order helices (not shown in Fig. 2 c) is also prohibited, since the pyridinium residues already occupy all available positions at both the top and the bottom of the double helix, making it impossible for additional charged residues to fit without overlapping. Strand 1 was synthesized in ten steps (overall yield: 5.8%) using iterative palladium-catalyzed Sonogashira cross-coupling reactions, as detailed in the Supplementary Information. The final product was isolated with either triflate ( 1·OTf ) or trifluoroacetate ( 1·TFA ) counterions. Both forms exhibited similar behavior by NMR spectroscopy (Supplementary Fig. S5), and only the results obtained for 1·TFA are discussed in detail below. The 1 H NMR spectrum of 1·TFA was first recorded in CD 3 CN, a moderately polar solvent that efficiently solvates the strand and does not promote folding. Under these conditions, 1 displayed sharp signals (Fig. 2 d) congruent with a discrete monomeric species with a hydrodynamic radius r H = 12.0 Å, estimated by 1 H diffusion-ordered spectroscopy (DOSY, Supplementary Fig. S2) using the Stokes-Einstein equation. Substantial spectral changes, indicative of duplex formation, were observed in D₂O (Fig. 2 e). The phenylene protons exhibited pronounced upfield shifts (e.g., Δδ = 0.3 ppm for proton k, Δδ = 0.5 ppm for n), consistent with increased shielding due to π-stacking interactions. Moreover, DOSY measurements conducted in D 2 O (Fig. 2 f) yielded a hydrodynamic radius r H = 8.4 Å, well aligned with the dimensions of the optimized DFT model for the double helix (ca. 3.9 Å in height and 9.5 Å in width). The reduction in hydrodynamic radius from the unfolded strand (12.0 Å) to the assembled duplex (8.4 Å) supported the formation of a more compact structure, closer to the spherical shape assumed in the Stokes-Einstein equation. Additional evidence for duplex formation came from 2D 1 H- 1 H rotating frame Overhauser effect spectroscopy (ROESY). The ROESY spectrum (Fig. 3 a) revealed multiple through-space ROE correlations between protons of stacked phenylene and pyridinium residues (e.g., p↔g p↔f ouside the helix, and k↔j and q↔j inside the helix). These cross-peaks would not be observable in a single strand (folded or not), as the corresponding protons would have been separated by distances of at least ~ 8 Å. A more detailed interpretation of the ROESY spectrum is provided in the Supplementary Information (Supplementary Fig. S8). The duplex exhibited remarkable stability in water. It retained its structural integrity upon dilution to concentrations as low as 0.5 mM (Supplementary Fig. S9) and resisted thermal denaturation at temperatures up to 348 K (Supplementary Fig. S10). As previously noted, duplex formation was independent of the counterions tested. However, subtle differences in the NMR spectra of helices derived from strands 1·TFA and 1·OTf (Supplementary Fig. S5) revealed distinct interactions between the duplex and its counterions. Triflate, being more chaotropic than trifluoroacetate, 45 is more likely to associate with the helix. This hypothesis was confirmed by titrating potassium triflate ( G1 , Fig. 3 b) into a 5 mM aqueous solution of 1·TFA . The addition of triflate caused relatively small shifts in the phenylene proton signals (Fig. 3 d and Supplementary Fig. S13), but more pronounced shifts in the pyridinium protons indicated preferential interaction with the polycationic outer rim of the helix. Assuming the formation of a 1:1 complex, analysis of the titration data yielded a relatively modest association constant of K ₐ = (75 ± 2) M⁻¹. We hypothesized that longer, more hydrophobic anions ( G2-G4 , Fig. 3 b) could bind more strongly by threading through the helix cavity. Indeed, addition of potassium perfluorobutanesulfonate ( G2 ) induced significant NMR shifts of both inner pyridinium and phenylene protons, consistent with the formation of an inclusion complex (Fig. 3 e and Supplementary Fig. S14). The enhanced binding affinity K ₐ = (5.7 ± 0.4) x 10 3 M⁻¹ was attributed to the increased hydrophobicity of G2 and its shape complementarity with the helix cavity. In contrast, the non-perfluorinated analogue sodium butanesulfonate G3 , which is less hydrophobic, showed no detectable interaction with the double helix (Supplementary Fig. S12). Finally, addition of sodium 4,4’-biphenyldisulfonate ( G4 ) resulted in substantial signal broadening, indicative of duplex dissociation, and ultimately led to precipitation (Supplementary Fig. S12). Overall, these results underscore the predictive accuracy of the sequence-to-structure relationship defined by inequality (3). The thermodynamic stability of the resulting double helix is exceptional, especially given the absence of inter-strand hydrogen bonding or metal coordination. Furthermore, the presence of a hydrophobic cavity within the helix enables the formation of host-guest complexes. One of these guests, perfluorobutanesulfonate G3 , is a persistent pollutant likely to be banned globally in the near future, 46 , 47 suggesting that oligo( m -phenylene ethynylene) helices could serve as platforms for capturing such contaminants from water. Controlling the assembly of multiple multistranded helices (dynamic behavior). Encouraged by this initial success, we sought to determine whether our approach could be extended to access more complex, dynamic systems. To this end, we designed strand 2 (Fig. 4 a), composed of h = 3 hydrophobic phenylene residues flanked by p = 2 polar pyridinium residues on each side. Given that l = 6 aromatic residues per turn for oligo( m -phenylene ethynylene) helices, the lower and upper bounds of inequality (3) are now l / h = 2 and l / p = 3, respectively. These values indicate that strand 2 can assemble into both double and triple helices. The corresponding energy diagram is showed in Fig. 4 b, and the equilibria involved are depicted in Fig. 4 c. This system is dynamic in nature, as it permits reversible switching between two distinct, thermodynamically stable helical states. Importantly, this dynamic behavior remains tightly constrained by the boundary conditions imposed by inequality (3). As a result, switching between double and triple helices occurs without any risk of forming the simpler single helix, a configuration typically favored in related systems, 25 – 27 , 48 , 49 but precluded here because n = 1 lies outside the predicted stability window. Strand 2 was synthesized in seven steps with an overall yield of 6.1% (see Supplementary Information), and its assembly was investigated using the protocol described above. In this case, the behavior of 2·OTf and 2·TFA differed markedly. This section focusses on 2·OTf ; the behavior of 2·TFA will be discussed in the following section. As expected, the NMR spectrum of 2·OTf in CD 3 CN (Fig. 4 d) exhibited sharp signals, consistent with a monomeric species ( r H = 10.8 Å, Supplementary Fig. S16). The signals progressively broadened upon gradual addition of D 2 O (Supplementary Fig. S20). This transformation culminated in pure D 2 O with the appearance of two new sets of resonances (Fig. 4 e), which were assigned to double helix ( 2 ) 2 (orange triangles) and triple helix ( 2 ) 3 (green circles) based on their chemical environments, hydrodynamic radii and ROE correlations. Double and triple helices could be individually characterized, because they exchange slowly on the NMR timescale and display well-resolved, distinguishable spectral features. In accordance with Le Chatelier’s principle, their relative abundance was found to be concentration dependent. Double helix ( 2 ) 2 was predominant at low concentrations (0.5 mM). Its resonances were upfield shifted relative to the unfolded strand and its hydrodynamic radius ( r H = 8.1 Å, Supplementary Fig. S22) closely matched that previously measured for double helix ( 1 ) 2 . The phenylene proton resonances of triple helix ( 2 ) 3 were shifted even further upfield (up to Dd = 0.5 ppm, compared to the double helix signals), as expected from a structure with an increased number of stacked aromatic surfaces. The triple helix was present only in trace amounts at low concentration (0.5 mM) but became the main species observable at 5 mM and above. Its hydrodynamic radius ( r H = 9.4 Å, Fig. 4 f) was larger than that of the double helix and in excellent agreement with the dimensions of the corresponding DFT model (ca. 5.8 Å height by 9.5 Å width). Two-dimensional ROESY spectra recorded at concentrations favoring either the double helix (0.5 mM, Fig. 5 a) or the triple helix (5 mM, Fig. 5 b) further supported our structural assignment. The two helices could be distinguished from the relative spatial arrangement of phenylene and pyridinium residues. For example, phenylene proton n showed through-space correlations with pyridinium proton c in the double helix, but with pyridinium protons f and g in the triple helix. Likewise, phenylene proton j correlated with pyridinium proton f in the double helix, but with pyridinium proton d in the triple helix. Additional diagnostic correlations are presented in Supplementary Figs. S27 and S28. These results further validate the predictive power of our sequence-to-structure model and demonstrate that bolaamphiphilic strands can be programmed to form multiple helical architectures in reversible exchange. Controlling the conformational switch between double and triple helices: role of concentration, temperature, and anions. Having established that strand 2·OTf assembles into double and triple helices in dynamic equilibrium, we examined how external parameters influenced this conformational switch. Specifically, we investigated the effects of concentration, temperature, and anionic guests on the relative populations of the two helical states. In general, triple helix ( 2 ) 3 was favored at higher concentrations and lower temperatures, while double helix ( 2 ) 2 predominated under dilute conditions and elevated temperatures. Varying both parameters thus provided a reliable means to control the composition of the system. For instance, at 10 mM, the triple helix remained stable across a broad temperature range (288–328 K, Supplementary Fig. S23). In contrast, at 3 mM, increasing the temperature from 288 K to 328 K induced a clear transition from the triple to the double helix (Supplementary Fig. S24). We next investigated whether the double-to-triple helix transition could be influenced by guest binding. As a starting point for this study, we compared the behaviors of strands 2·TFA and 2·OTf , which differ only in their counterions. The ¹H NMR spectrum of 2·TFA at 5 mM in D₂O (Fig. 5 c) displayed upfield-shifted resonances characteristic of the triple helix, but these signals were significantly broader than those previously observed for 2·OTf (Fig. 4 e). This difference suggested that the more chaotropic triflate occupies, at least partially, the cavity of the triple helix, thereby restricting its conformational motion. This interpretation was supported by the observation that adding potassium triflate G1 to a 5 mM aqueous solution of 2·TFA led to progressive sharpening and shifting of the triple helix resonances (Supplementary Fig. S30). The shifts were more pronounced for inner phenylene protons, as expected if triflate binds within the cavity. Fitting the titration data using a 1:1 binding model yielded an association constant of K ₐ = (9.8 ± 0.7) x 10 2 M⁻¹, approximately one order of magnitude higher than that previously measured for ( 1 ) 2 . This increased affinity is attributed to the longer and more hydrophobic internal cavity of the triple helix ( 2 )₃, which provides a more favorable environment for guest encapsulation. Similar signal sharpening was observed upon addition of potassium perfluorobutanesulfonate ( G2 , Supplementary Fig. S31) and sodium 4,4’-biphenyldisulfonate ( G4 , Supplementary Figs. S32 and S33), indicating the formation of inclusion complexes ( 2 )₃⊃ G2 and ( 2 )₃⊃ G4 , respectively. Although the broad resonances at intermediate titration points precluded accurate determination of association constants for both guests, the importance of hydrophobicity in guest binding was highlighted once again by the lack of measurable interaction between sodium butanesulfonate G3 and the triple helix (Supplementary Fig. S29). Concentration- and temperature-dependent NMR spectra recorded after addition of G1 , G2 and G4 (Supplementary Figs. S29-S39) demonstrated a clean transition between the double and triple helical states in the presence of these guests. A representative example of these experiments, obtained after addition of G2 , is shown in Figs. 5 d-e. Comparisons between the effects of different anions on the switching behavior should be made with caution, as they depend on both the association constants with the double and triple helix, and the number of guest equivalents added. Nevertheless, the data suggest that more hydrophobic guests, expected to bind more strongly the triple helix, stabilize it and delay its transition to the double helix both upon dilution and heating. The most stable complexes, ( 2 )₃⊃ G2 and ( 2 )₃⊃ G4 , were modeled by DFT at the BP86-D4-COSMO(water)/def2-TZVP level of theory (Figs. 5 f-g), confirming a good geometric fit of these guests within the cavity of the triple helix. In the case of G4 , p-p stacking and electrostatic attraction between the aryl sulfonate moieties and the pyridinium units induces dynamic distortions of the triple helix, consistent with the slightly broader NMR signals obtained for the corresponding complex. Taken together, these results demonstrate that three external parameters (concentration, temperature, and the presence of suitable anionic guests) can be used to reversibly control the conformational equilibrium between double and triple helices. Importantly, the switching process remained tightly controlled and cleanly operable under all tested conditions, with no detectable formation of the undesired single helices or strand unfolding. Conclusion Two bolaamphiphilic oligo( m -phenylene ethynylene) strands were synthesized to demonstrate that the relative lengths of hydrophobic and charged segments dictate the formation of distinct helical architectures. As predicted, strand 1 assembled exclusively into a stable double helix, whereas strand 2 formed both double and triple helices in dynamic equilibrium. The latter system enabled clean, reversible switching between the permitted helical states in response to concentration, temperature, or binding of specific amphiphilic guests. This study establishes a robust strategy for controlling both the assembly and dynamic behavior of multistranded helices through the primary sequence of synthetic strands. A key strength of this approach lies in its minimalism: precise structural control was achieved using only two building blocks, hydrophobic phenylene and charged pyridinium residues. This simplicity renders the approach modular and potentially extensible to alternative sequence patterns (including non-bolaamphiphilic ones) and other aromatic foldamer scaffolds. Moreover, the oligo( m -phenylene ethynylene) backbone is amenable to chemical modification, 38 , 41 allowing for the integration of catalytic, recognition, or sensing functions within structurally programmed helices. More broadly, the ability to encode multiple, switchable helical states within a single strand introduces a new level of control in the design of adaptive molecular systems. By establishing a clear and predictable sequence-to-structure relationship, this work may enable the construction of complex, programmable supramolecular architectures with responsive functionalities. Declarations Data availability The data supporting the findings of this study are available in the manuscript, in the Supplementary Information, or from the lead contact. Acknowledgements This work was supported by the Research Council of Finland (grant 357271) and the University of Jyväskylä. A.F. and R.M.G. are grateful for project PID2023-148453NB-I00 funded by the Ministerio de Ciencia, Innovación y Universidades of Spain MCIU/AEI/10.13039/501100011033 and FEDER, UE. Author contributions D.D., R.A. and P.V. performed chemical synthesis. D.D. and P.P. performed NMR analysis. A.F. and R.M.G. performed computational work. F.B.L.C. conceptualized and supervised the project. The paper was written through contributions from all authors, and all authors have given approval to the final version of the paper. Additional information Supplementary Information contains synthetic procedures, characterization data and theoretical calculations. 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J Am Chem Soc 147:22768–22777 Berl V, Huc I, Khoury RG, Krische MJ, Lehn J-M (2000) Interconversion of single and double helices formed from synthetic molecular strands. Nature 407:720–723 Jiang H, Maurizot V, Huc I (2004) Double versus single helical structures of oligopyridine-dicarboxamide strands. Part 1: Effect of oligomer length. Tetrahedron 60:10029–10038 Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryInformation.pdf Supplementary Information for “Programmable Assembly of Multistranded Helices in Water” Cite Share Download PDF Status: Published Journal Publication published 11 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7441733","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":509008662,"identity":"b833e51c-ed1f-4068-a33d-f596be8389b2","order_by":0,"name":"Fabien Cougnon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIie3PsWoCMRjA8e8I5JbYrAkOvkJqV8FXuaPQSW85cGqPAyFTi6sHPoRjxxwHulRnR12cpMStHQpNLCotRB0L5j8k5CM/QgB8vn9YmBNQZo8gPMyQEmal2EGI2hN0mOHIEi5PEPhDiBXAcxcJZ6XavkJiSKC38imh9Wed1iSwGxchSVQWb5AagnghpykfzcZ3lrj+0oaOqMyFOEeAUU1O4vGiuyOZ8/t0I6qv36SzPPkKYeaV4EgeLYEz5D0qXyRLMQr6fDhXcTF8ELejOXMT2q30p2wlNOyXWveyeMDu12LTa7FG7jA/McAQ2CuVPeEmMZNLy+yCVh8XA5/P57uCvgH051GovNGIFQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4487-8707","institution":"University of Jyväskylä","correspondingAuthor":true,"prefix":"","firstName":"Fabien","middleName":"","lastName":"Cougnon","suffix":""},{"id":509008663,"identity":"37c0724e-e2c0-4278-b245-8f8486f2f10d","order_by":1,"name":"Dimitri Delcourt","email":"","orcid":"https://orcid.org/0000-0001-6012-4791","institution":"University of Jyväskylä","correspondingAuthor":false,"prefix":"","firstName":"Dimitri","middleName":"","lastName":"Delcourt","suffix":""},{"id":509008664,"identity":"45874c64-da86-42c1-b848-45d16df6268a","order_by":2,"name":"Reguram Arumugaperumal","email":"","orcid":"","institution":"University of Jyväskylä","correspondingAuthor":false,"prefix":"","firstName":"Reguram","middleName":"","lastName":"Arumugaperumal","suffix":""},{"id":509008665,"identity":"a05e0e50-fa48-46eb-b90d-9bb7856dafb0","order_by":3,"name":"Prachi Verma","email":"","orcid":"","institution":"University of Jyväskylä","correspondingAuthor":false,"prefix":"","firstName":"Prachi","middleName":"","lastName":"Verma","suffix":""},{"id":509008666,"identity":"ef66c297-e363-4544-9d15-1ae483daa9e7","order_by":4,"name":"Perttu Permi","email":"","orcid":"https://orcid.org/0000-0002-6281-1138","institution":"University of Jyvaskyla","correspondingAuthor":false,"prefix":"","firstName":"Perttu","middleName":"","lastName":"Permi","suffix":""},{"id":509008667,"identity":"196fd90f-b667-4535-9b03-b439798a3163","order_by":5,"name":"Rosa Gomila","email":"","orcid":"","institution":"Universitat de les Illes Balears","correspondingAuthor":false,"prefix":"","firstName":"Rosa","middleName":"","lastName":"Gomila","suffix":""},{"id":509008668,"identity":"9f6e7c26-62dc-45ce-808b-6c81c36c7b92","order_by":6,"name":"Antonio Frontera","email":"","orcid":"https://orcid.org/0000-0001-7840-2139","institution":"Universitat de les Illes Balears","correspondingAuthor":false,"prefix":"","firstName":"Antonio","middleName":"","lastName":"Frontera","suffix":""}],"badges":[],"createdAt":"2025-08-23 14:00:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7441733/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7441733/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-67227-0","type":"published","date":"2025-12-11T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90891515,"identity":"8ab48a10-618c-4596-9050-e8f052035f18","added_by":"auto","created_at":"2025-09-09 11:11:24","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":47885,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSequence-encoded assembly of multistranded helices from bolaamphiphilic molecular strands in water. a, \u003c/strong\u003eSchematic representation of a bolaamphiphilic strand assembling into \u003cem\u003en\u003c/em\u003e-stranded helices in water. The hydrophobic core length is denoted by \u003cem\u003eh\u003c/em\u003e, the length of the polar, permanently charged termini by \u003cem\u003ep\u003c/em\u003e, and the length of strand require to complete one helical turn by \u003cem\u003el\u003c/em\u003e. \u003cstrong\u003eb,\u003c/strong\u003e Cutaway view of a helix illustrating how geometric constraints, set by the primary sequence of the strand, determine the type of helix that forms.\u003c/p\u003e","description":"","filename":"image1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7441733/v1/e189cd598ce34140e989e363.jpg"},{"id":90891516,"identity":"5b3342d9-885a-4961-9efd-acc7a4cec6b4","added_by":"auto","created_at":"2025-09-09 11:11:24","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":107883,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProgrammable assembly of a double helix in aqueous solution. a, \u003c/strong\u003eChemical structure of strand \u003cstrong\u003e1·TFA\u003c/strong\u003e. \u003cstrong\u003eb,\u003c/strong\u003e Energy diagram showing that the double helix is the only thermodynamically favored state in water. \u003cstrong\u003ec,\u003c/strong\u003e Equilibria involved in the formation of double helix (\u003cstrong\u003e1\u003c/strong\u003e)\u003csub\u003e2\u003c/sub\u003e. Double helix (\u003cstrong\u003e1\u003c/strong\u003e)\u003csub\u003e2\u003c/sub\u003e was modeled using a BP86-D4-COSMO(water)/def2-TZVP level of theory. \u003cstrong\u003ed,\u003c/strong\u003e \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of unfolded strand \u003cstrong\u003e1·TFA\u003c/strong\u003e in CD\u003csub\u003e3\u003c/sub\u003eCN (500 MHz, 298 K). \u003cstrong\u003ee,\u003c/strong\u003e \u003csup\u003e1\u003c/sup\u003eH NMR and \u003cstrong\u003ef,\u003c/strong\u003e DOSY spectra of double helix (\u003cstrong\u003e1\u003c/strong\u003e)\u003csub\u003e2\u003c/sub\u003e\u003cstrong\u003e·TFA\u003c/strong\u003e in D\u003csub\u003e2\u003c/sub\u003eO (10 mM, 500 MHz, 298 K).\u003c/p\u003e","description":"","filename":"image2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7441733/v1/152a0f7715026c4f91760100.jpg"},{"id":90891517,"identity":"002731ca-0fac-4c52-84b4-7ceab1ec29a3","added_by":"auto","created_at":"2025-09-09 11:11:24","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":80780,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural characterization and host-guest chemistry of double helix (1)\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e. a,\u003c/strong\u003e Partial 2D \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH ROESY NMR spectrum of double helix (\u003cstrong\u003e1\u003c/strong\u003e)\u003csub\u003e2\u003c/sub\u003e\u003cstrong\u003e·TFA\u003c/strong\u003e in D\u003csub\u003e2\u003c/sub\u003eO (10 mM, 500 MHz, 278 K, 100 ms mixing time), showing key through-space correlations between protons of stacked pyridinium (blue) and phenylene (gray) residues. \u003cstrong\u003eb,\u003c/strong\u003e Chemical structure of hydrophobic sulfonates \u003cstrong\u003eG1\u003c/strong\u003e-\u003cstrong\u003eG4\u003c/strong\u003e. \u003cstrong\u003ec-e,\u003c/strong\u003e Partial \u003csup\u003e1\u003c/sup\u003eH NMR spectra of triple helix \u003cstrong\u003e2·TFA\u003c/strong\u003e in D\u003csub\u003e2\u003c/sub\u003eO (5 mM in D\u003csub\u003e2\u003c/sub\u003eO, 500 MHz, 298 K): \u003cstrong\u003ec,\u003c/strong\u003e free host, \u003cstrong\u003ed,\u003c/strong\u003e after addition of 6.0 equiv. of\u003cstrong\u003e G1\u003c/strong\u003e, \u003cstrong\u003ee,\u003c/strong\u003e after addition of 3.0 equiv. of \u003cstrong\u003eG2\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7441733/v1/d4e8fa7df4abdcb380f67155.jpg"},{"id":90891520,"identity":"494afc1d-9496-4f65-8eb7-06643bea2f9f","added_by":"auto","created_at":"2025-09-09 11:11:24","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":132477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProgrammable assembly of double and triple helices in dynamic exchange in aqueous solution. a, \u003c/strong\u003eChemical structure of strand \u003cstrong\u003e2·OTf\u003c/strong\u003e. \u003cstrong\u003eb,\u003c/strong\u003e Energy diagram indicating that the double and triple helices are the only thermodynamically favored state in water. \u003cstrong\u003ec,\u003c/strong\u003e Equilibria governing the formation of double helix (\u003cstrong\u003e2\u003c/strong\u003e)\u003csub\u003e2\u003c/sub\u003e and\u003csub\u003e \u003c/sub\u003etriple helix (\u003cstrong\u003e2\u003c/strong\u003e)\u003csub\u003e3\u003c/sub\u003e. Double helix (\u003cstrong\u003e2\u003c/strong\u003e)\u003csub\u003e2\u003c/sub\u003e and triple helix (\u003cstrong\u003e2\u003c/strong\u003e)\u003csub\u003e3\u003c/sub\u003e were both modeled using a BP86-D4-COSMO(water)/def2-TZVP level of theory. \u003cstrong\u003ed,\u003c/strong\u003e \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of unfolded strand \u003cstrong\u003e2·OTf\u003c/strong\u003e in CD\u003csub\u003e3\u003c/sub\u003eCN (500 MHz, 298 K). \u003cstrong\u003ee, \u003c/strong\u003eConcentration-dependent \u003csup\u003e1\u003c/sup\u003eH NMR spectra showing the formation of double helix (\u003cstrong\u003e2\u003c/strong\u003e)\u003csub\u003e2\u003c/sub\u003e\u003cstrong\u003e·OTf\u003c/strong\u003e and\u003csub\u003e \u003c/sub\u003etriple helix (\u003cstrong\u003e2\u003c/strong\u003e)\u003csub\u003e3\u003c/sub\u003e\u003cstrong\u003e·OTf\u003c/strong\u003e in D\u003csub\u003e2\u003c/sub\u003eO (800 MHz, 288 K). \u003cstrong\u003ef,\u003c/strong\u003e DOSY spectrum of triple helix (\u003cstrong\u003e2\u003c/strong\u003e)\u003csub\u003e2\u003c/sub\u003e\u003cstrong\u003e·OTf\u003c/strong\u003e in D\u003csub\u003e2\u003c/sub\u003eO (10 mM, 800 MHz, 288 K).\u003c/p\u003e","description":"","filename":"image4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7441733/v1/2d69501bfebbbaab00be3b96.jpg"},{"id":90891519,"identity":"9a998eb6-059d-4930-abf5-3c6e9d05f0b8","added_by":"auto","created_at":"2025-09-09 11:11:24","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":114674,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization and dynamic behavior of double helix (2)\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and triple helix (2)\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e. a,b, \u003c/strong\u003ePartial 2D \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH ROESY NMR spectra of \u003cstrong\u003ea,\u003c/strong\u003e double helix (\u003cstrong\u003e2\u003c/strong\u003e)\u003csub\u003e2\u003c/sub\u003e\u003cstrong\u003e·OTf\u003c/strong\u003e in D\u003csub\u003e2\u003c/sub\u003eO (0.5 mM) and \u003cstrong\u003eb,\u003c/strong\u003e triple helix (\u003cstrong\u003e2\u003c/strong\u003e)\u003csub\u003e3\u003c/sub\u003e\u003cstrong\u003e·OTf\u003c/strong\u003e (10 mM) in D\u003csub\u003e2\u003c/sub\u003eO (800 MHz, 288 K, 200 ms mixing time), highlighting key through-space correlations between protons of stacked pyridinium (blue) and phenylene (gray) residues. \u003cstrong\u003ec,\u003c/strong\u003e \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of triple helix \u003cstrong\u003e2·TFA\u003c/strong\u003e in D\u003csub\u003e2\u003c/sub\u003eO (500 MHz, 298 K). \u003cstrong\u003ed, \u003c/strong\u003eConcentration-dependent \u003csup\u003e1\u003c/sup\u003eH NMR spectra of \u003cstrong\u003e2·TFA\u003c/strong\u003e (5 to 0.5 mM) in D\u003csub\u003e2\u003c/sub\u003eO (500 MHz, 298 K) in the presence of 5.0 equiv. of potassium perfluorobutanesulfonate \u003cstrong\u003eG2\u003c/strong\u003e. \u003cstrong\u003ee,\u003c/strong\u003e Variable temperature \u003csup\u003e1\u003c/sup\u003eH NMR spectra of \u003cstrong\u003e2·TFA\u003c/strong\u003e recorded between 278 K and 348 K in D\u003csub\u003e2\u003c/sub\u003eO (0.5 mM, 500 MHz) in the presence of 5.0 equiv. of potassium perfluorobutanesulfonate \u003cstrong\u003eG2\u003c/strong\u003e. Signals corresponding to the double and triple helices are labelled with orange triangles and green circles, respectively. \u003cstrong\u003ef,\u003c/strong\u003e DFT minimized model of (\u003cstrong\u003e2\u003c/strong\u003e)₃⊃\u003cstrong\u003eG2 \u003c/strong\u003e[BP86-D4-COSMO(water)/def2-TZVP]. \u003cstrong\u003eg,\u003c/strong\u003e DFT minimized model of (\u003cstrong\u003e2\u003c/strong\u003e)₃⊃\u003cstrong\u003eG4 \u003c/strong\u003e[BP86-D4-COSMO(water)/def2-TZVP].\u003c/p\u003e","description":"","filename":"image5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7441733/v1/f4897d72eaecbfbc24970bd1.jpg"},{"id":98046056,"identity":"51318919-8b77-4235-833d-38a2cf01fe14","added_by":"auto","created_at":"2025-12-12 08:10:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1391900,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7441733/v1/e4543e7a-1180-42d0-81f4-4c9a50fd2ae6.pdf"},{"id":90891521,"identity":"69c79ab7-efce-4e61-b30f-a3296127ec86","added_by":"auto","created_at":"2025-09-09 11:11:24","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13712174,"visible":true,"origin":"","legend":"Supplementary Information for \u0026#x201C;Programmable Assembly of Multistranded Helices in Water\u0026#x201D;","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7441733/v1/bac9bd282f2cb061c2c7b75e.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Programmable Assembly of Multistranded Helices in Water","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe primary sequence of proteins and nucleic acids defines a landscape of energetically accessible conformational states, enabling these biomolecules to undergo controlled structural transitions in response to external cues.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e The primary sequence of DNA, for example, determines its ability to adopt a variety of higher-order structures beyond the canonical right-handed B-form, including left-handed double helices (Z-form), triple helices (H-form), and G-quadruplexes.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Transitions between these different states play essential roles in key cellular processes like gene expression, replication, and repair.\u003c/p\u003e\u003cp\u003eSignificant efforts have been devoted to developing synthetic analogs that mimic the structural and functional complexity of biomolecules. Synthetic multistranded helices have emerged as promising biomimetic platforms for applications such as molecular recognition,\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8 CR9\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e switching,\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15 CR16\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e and self-replication.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Their assembly typically relies on non-covalent interactions, including hydrogen bonding,\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e π\u0026ndash;π stacking,\u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24 CR25 CR26 CR27 CR28\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and metal coordination.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Despite the wide range of strategies available to control helix formation, general methods for encoding both structural organization and dynamic responsiveness directly into the primary sequence of synthetic strands have yet to be established. In a landmark study, the Flood group demonstrated sequence control over single- to double-helix transitions in aryl-triazole foldamers.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e However, extending this approach beyond that specific system remains an open challenge. Developing broadly applicable strategies that couple sequence information to both structure and dynamics would unlock a new generation of synthetic systems with biomolecule-like functions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHere we present an approach for encoding, within the primary sequence of synthetic strands, all the information necessary to control both the assembly and dynamic behavior of multistranded helices. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea shows the general structure of a bolaamphiphilic strand, consisting of a central hydrophobic core flanked by two permanently charged, polar termini. Let \u003cem\u003eh\u003c/em\u003e and \u003cem\u003ep\u003c/em\u003e denote the lengths of the hydrophobic and polar segments, respectively. In aqueous environments, such bolaamphiphilic sequences promote the self-assembly of \u003cem\u003en-\u003c/em\u003estranded (where \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1, 2, 3, \u0026hellip;) helices, which minimize exposure of the hydrophobic surface to water by positioning the polar segments at the top and bottom of the hydrophobic core. However, these helices form only if adjacent charged segments do not stack directly above one another, as it would generate destabilizing electrostatic repulsion.\u003c/p\u003e\u003cp\u003eElectrostatic repulsion may arise between charged components situated on the same face of the helix, or between those located on opposite faces (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Repulsion between charged segments located on the same face (e.g., the upper rim) can be avoided if the combined length of these charged segments (\u003cem\u003en\u0026middot;p\u003c/em\u003e) does not exceed the length of one full helical turn. Defining \u003cem\u003el\u003c/em\u003e as the length of strand required to complete one helical turn, this condition can be expressed as:\u003c/p\u003e\u003cp\u003e\u003cb\u003en\u0026middot;p\u0026thinsp;\u0026le;\u0026thinsp;l\u003c/b\u003e (1)\u003c/p\u003e\u003cp\u003eBy symmetry, this same constraint applies to the charged segments located at the lower rim of the helix.\u003c/p\u003e\u003cp\u003eRepulsion between charged segments located on opposite faces of the helix can be avoided if they are separated by a full hydrophobic layer; that is, if the combined length of hydrophobic components located at the center of the structure (\u003cem\u003en\u0026middot;h\u003c/em\u003e) is greater or equal to \u003cem\u003el\u003c/em\u003e:\u003c/p\u003e\u003cp\u003e\u003cb\u003en\u0026middot;h\u0026thinsp;\u0026ge;\u0026thinsp;l\u003c/b\u003e (2)\u003c/p\u003e\u003cp\u003eCombining inequalities (1) and (2) yields a single inequality that defines the permissible range for the number of strands \u003cem\u003en\u003c/em\u003e:\u003c/p\u003e\u003cp\u003e\u003cb\u003el/h\u0026thinsp;\u0026le;\u0026thinsp;n\u0026thinsp;\u0026le;\u0026thinsp;l/p\u003c/b\u003e (3)\u003c/p\u003e\u003cp\u003eThis inequality establishes a direct sequence-to-structure relationship: the primary sequence of the strand (characterized by parameters \u003cem\u003eh\u003c/em\u003e, \u003cem\u003ep\u003c/em\u003e, and \u003cem\u003el\u003c/em\u003e) sets the geometric window within which \u003cem\u003en\u003c/em\u003e-stranded helices are stable. When \u003cem\u003en\u003c/em\u003e falls within this window, helix formation is thermodynamically favored, as it reduces hydrophobic exposure without introducing repulsive interactions. Outside this range, helix formation is disfavored due to unavoidable electrostatic destabilization. Inequality (3) can thus be used to parametrize strands capable of forming specific \u003cem\u003en\u003c/em\u003e-stranded helices.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eIn this article, we sought to experimentally test the predictions of the model described above. Oligo(\u003cem\u003em\u003c/em\u003e-phenylene ethynylene)s were selected as a suitable scaffold, because their conformational behavior is well documented.\u003csup\u003e\u003cspan additionalcitationids=\"CR33 CR34 CR35 CR36\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Moore demonstrated their strong tendency to fold into single helices several decades ago.\u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e The folding process is driven by solvophobic effects and can be promoted by polar solvents such as water.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e More recently, Berryman showed that oligo(\u003cem\u003em\u003c/em\u003e-phenylene ethynylene)s can also form higher-order helices.\u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e Building on these foundations, we reengineered the oligo(\u003cem\u003em\u003c/em\u003e-phenylene ethynylene) scaffold by introducing hydrophobic and polar segments of variable lengths, and assessed whether the resulting sequences predictably yielded specific types of multistranded helices.\u003c/p\u003e\u003cp\u003e\u003cb\u003eControlling the assembly of a single type of multistranded helix (static behavior).\u003c/b\u003e We first designed strand \u003cb\u003e1\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), composed of \u003cem\u003eh\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3 hydrophobic phenylene residues and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3 polar pyridinium residues. Since oligo(\u003cem\u003em\u003c/em\u003e-phenylene ethynylene) helices require \u003cem\u003el\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 aromatic residues per turn,\u003csup\u003e\u003cspan additionalcitationids=\"CR39 CR40 CR41 CR42 CR43\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e the lower and upper bounds of inequality (3) are \u003cem\u003el\u003c/em\u003e/\u003cem\u003eh\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2 and \u003cem\u003el\u003c/em\u003e/\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2, respectively. Because \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2 is the only integer that satisfies this condition, strand \u003cb\u003e1\u003c/b\u003e is predicted to exclusively assemble into a double helix. This situation is illustrated schematically in the free energy diagram shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, where the double helix represents the only thermodynamically stable state (DG\u0026deg; \u0026lt; 0).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe equilibria involved in the assembly of the double helix are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec. In agreement with our predictions, DFT calculations performed at a BP86-D4-COSMO(water)/def2-TZVP level of theory showed that the formation of a double helix allows the outer pyridiniums to effectively shield the hydrophobic phenylenes from water. Single helix formation is disfavored, as it would necessarily bring charged pyridiniums in close contact, resulting in destabilizing electrostatic repulsion. The assembly of higher-order helices (not shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) is also prohibited, since the pyridinium residues already occupy all available positions at both the top and the bottom of the double helix, making it impossible for additional charged residues to fit without overlapping.\u003c/p\u003e\u003cp\u003eStrand \u003cb\u003e1\u003c/b\u003e was synthesized in ten steps (overall yield: 5.8%) using iterative palladium-catalyzed Sonogashira cross-coupling reactions, as detailed in the Supplementary Information. The final product was isolated with either triflate (\u003cb\u003e1\u0026middot;OTf\u003c/b\u003e) or trifluoroacetate (\u003cb\u003e1\u0026middot;TFA\u003c/b\u003e) counterions. Both forms exhibited similar behavior by NMR spectroscopy (Supplementary Fig. S5), and only the results obtained for \u003cb\u003e1\u0026middot;TFA\u003c/b\u003e are discussed in detail below.\u003c/p\u003e\u003cp\u003eThe \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR spectrum of \u003cb\u003e1\u0026middot;TFA\u003c/b\u003e was first recorded in CD\u003csub\u003e3\u003c/sub\u003eCN, a moderately polar solvent that efficiently solvates the strand and does not promote folding. Under these conditions, \u003cb\u003e1\u003c/b\u003e displayed sharp signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed) congruent with a discrete monomeric species with a hydrodynamic radius \u003cem\u003er\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e = 12.0 \u0026Aring;, estimated by \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH diffusion-ordered spectroscopy (DOSY, Supplementary Fig. S2) using the Stokes-Einstein equation.\u003c/p\u003e\u003cp\u003eSubstantial spectral changes, indicative of duplex formation, were observed in D₂O (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). The phenylene protons exhibited pronounced upfield shifts (e.g., Δδ\u0026thinsp;=\u0026thinsp;0.3 ppm for proton k, Δδ\u0026thinsp;=\u0026thinsp;0.5 ppm for n), consistent with increased shielding due to π-stacking interactions. Moreover, DOSY measurements conducted in D\u003csub\u003e2\u003c/sub\u003eO (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef) yielded a hydrodynamic radius \u003cem\u003er\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e = 8.4 \u0026Aring;, well aligned with the dimensions of the optimized DFT model for the double helix (ca. 3.9 \u0026Aring; in height and 9.5 \u0026Aring; in width). The reduction in hydrodynamic radius from the unfolded strand (12.0 \u0026Aring;) to the assembled duplex (8.4 \u0026Aring;) supported the formation of a more compact structure, closer to the spherical shape assumed in the Stokes-Einstein equation.\u003c/p\u003e\u003cp\u003eAdditional evidence for duplex formation came from 2D \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH-\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH rotating frame Overhauser effect spectroscopy (ROESY). The ROESY spectrum (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea) revealed multiple through-space ROE correlations between protons of stacked phenylene and pyridinium residues (e.g., p\u0026harr;g p\u0026harr;f ouside the helix, and k\u0026harr;j and q\u0026harr;j inside the helix). These cross-peaks would not be observable in a single strand (folded or not), as the corresponding protons would have been separated by distances of at least\u0026thinsp;~\u0026thinsp;8 \u0026Aring;. A more detailed interpretation of the ROESY spectrum is provided in the Supplementary Information (Supplementary Fig. S8).\u003c/p\u003e\u003cp\u003eThe duplex exhibited remarkable stability in water. It retained its structural integrity upon dilution to concentrations as low as 0.5 mM (Supplementary Fig. S9) and resisted thermal denaturation at temperatures up to 348 K (Supplementary Fig. S10). As previously noted, duplex formation was independent of the counterions tested. However, subtle differences in the NMR spectra of helices derived from strands \u003cb\u003e1\u0026middot;TFA\u003c/b\u003e and \u003cb\u003e1\u0026middot;OTf\u003c/b\u003e (Supplementary Fig. S5) revealed distinct interactions between the duplex and its counterions. Triflate, being more chaotropic than trifluoroacetate,\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e is more likely to associate with the helix. This hypothesis was confirmed by titrating potassium triflate (\u003cb\u003eG1\u003c/b\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) into a 5 mM aqueous solution of \u003cb\u003e1\u0026middot;TFA\u003c/b\u003e. The addition of triflate caused relatively small shifts in the phenylene proton signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and Supplementary Fig. S13), but more pronounced shifts in the pyridinium protons indicated preferential interaction with the polycationic outer rim of the helix. Assuming the formation of a 1:1 complex, analysis of the titration data yielded a relatively modest association constant of \u003cem\u003eK\u003c/em\u003eₐ = (75\u0026thinsp;\u0026plusmn;\u0026thinsp;2) M⁻\u0026sup1;.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe hypothesized that longer, more hydrophobic anions (\u003cb\u003eG2-G4\u003c/b\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) could bind more strongly by threading through the helix cavity. Indeed, addition of potassium perfluorobutanesulfonate (\u003cb\u003eG2\u003c/b\u003e) induced significant NMR shifts of both inner pyridinium and phenylene protons, consistent with the formation of an inclusion complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee and Supplementary Fig. S14). The enhanced binding affinity \u003cem\u003eK\u003c/em\u003eₐ = (5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4) x 10\u003csup\u003e3\u003c/sup\u003e M⁻\u0026sup1; was attributed to the increased hydrophobicity of \u003cb\u003eG2\u003c/b\u003e and its shape complementarity with the helix cavity. In contrast, the non-perfluorinated analogue sodium butanesulfonate \u003cb\u003eG3\u003c/b\u003e, which is less hydrophobic, showed no detectable interaction with the double helix (Supplementary Fig. S12). Finally, addition of sodium 4,4\u0026rsquo;-biphenyldisulfonate (\u003cb\u003eG4\u003c/b\u003e) resulted in substantial signal broadening, indicative of duplex dissociation, and ultimately led to precipitation (Supplementary Fig. S12).\u003c/p\u003e\u003cp\u003eOverall, these results underscore the predictive accuracy of the sequence-to-structure relationship defined by inequality (3). The thermodynamic stability of the resulting double helix is exceptional, especially given the absence of inter-strand hydrogen bonding or metal coordination. Furthermore, the presence of a hydrophobic cavity within the helix enables the formation of host-guest complexes. One of these guests, perfluorobutanesulfonate \u003cb\u003eG3\u003c/b\u003e, is a persistent pollutant likely to be banned globally in the near future,\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e suggesting that oligo(\u003cem\u003em\u003c/em\u003e-phenylene ethynylene) helices could serve as platforms for capturing such contaminants from water.\u003c/p\u003e\u003cp\u003e\u003cb\u003eControlling the assembly of multiple multistranded helices (dynamic behavior).\u003c/b\u003e Encouraged by this initial success, we sought to determine whether our approach could be extended to access more complex, dynamic systems. To this end, we designed strand \u003cb\u003e2\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), composed of \u003cem\u003eh\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3 hydrophobic phenylene residues flanked by \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2 polar pyridinium residues on each side. Given that \u003cem\u003el\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 aromatic residues per turn for oligo(\u003cem\u003em\u003c/em\u003e-phenylene ethynylene) helices, the lower and upper bounds of inequality (3) are now \u003cem\u003el\u003c/em\u003e/\u003cem\u003eh\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2 and \u003cem\u003el\u003c/em\u003e/\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3, respectively. These values indicate that strand \u003cb\u003e2\u003c/b\u003e can assemble into both double and triple helices. The corresponding energy diagram is showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, and the equilibria involved are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec.\u003c/p\u003e\u003cp\u003eThis system is dynamic in nature, as it permits reversible switching between two distinct, thermodynamically stable helical states. Importantly, this dynamic behavior remains tightly constrained by the boundary conditions imposed by inequality (3). As a result, switching between double and triple helices occurs without any risk of forming the simpler single helix, a configuration typically favored in related systems,\u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e but precluded here because \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1 lies outside the predicted stability window.\u003c/p\u003e\u003cp\u003eStrand \u003cb\u003e2\u003c/b\u003e was synthesized in seven steps with an overall yield of 6.1% (see Supplementary Information), and its assembly was investigated using the protocol described above. In this case, the behavior of \u003cb\u003e2\u0026middot;OTf\u003c/b\u003e and \u003cb\u003e2\u0026middot;TFA\u003c/b\u003e differed markedly. This section focusses on \u003cb\u003e2\u0026middot;OTf\u003c/b\u003e; the behavior of \u003cb\u003e2\u0026middot;TFA\u003c/b\u003e will be discussed in the following section.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs expected, the NMR spectrum of \u003cb\u003e2\u0026middot;OTf\u003c/b\u003e in CD\u003csub\u003e3\u003c/sub\u003eCN (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) exhibited sharp signals, consistent with a monomeric species (\u003cem\u003er\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e = 10.8 \u0026Aring;, Supplementary Fig. S16). The signals progressively broadened upon gradual addition of D\u003csub\u003e2\u003c/sub\u003eO (Supplementary Fig. S20). This transformation culminated in pure D\u003csub\u003e2\u003c/sub\u003eO with the appearance of two new sets of resonances (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), which were assigned to double helix (\u003cb\u003e2\u003c/b\u003e)\u003csub\u003e2\u003c/sub\u003e (orange triangles) and triple helix (\u003cb\u003e2\u003c/b\u003e)\u003csub\u003e3\u003c/sub\u003e (green circles) based on their chemical environments, hydrodynamic radii and ROE correlations.\u003c/p\u003e\u003cp\u003eDouble and triple helices could be individually characterized, because they exchange slowly on the NMR timescale and display well-resolved, distinguishable spectral features. In accordance with Le Chatelier\u0026rsquo;s principle, their relative abundance was found to be concentration dependent. Double helix (\u003cb\u003e2\u003c/b\u003e)\u003csub\u003e2\u003c/sub\u003e was predominant at low concentrations (0.5 mM). Its resonances were upfield shifted relative to the unfolded strand and its hydrodynamic radius (\u003cem\u003er\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e = 8.1 \u0026Aring;, Supplementary Fig. S22) closely matched that previously measured for double helix (\u003cb\u003e1\u003c/b\u003e)\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eThe phenylene proton resonances of triple helix (\u003cb\u003e2\u003c/b\u003e)\u003csub\u003e3\u003c/sub\u003e were shifted even further upfield (up to Dd\u0026thinsp;=\u0026thinsp;0.5 ppm, compared to the double helix signals), as expected from a structure with an increased number of stacked aromatic surfaces. The triple helix was present only in trace amounts at low concentration (0.5 mM) but became the main species observable at 5 mM and above. Its hydrodynamic radius (\u003cem\u003er\u003c/em\u003e\u003csub\u003eH\u003c/sub\u003e = 9.4 \u0026Aring;, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef) was larger than that of the double helix and in excellent agreement with the dimensions of the corresponding DFT model (ca. 5.8 \u0026Aring; height by 9.5 \u0026Aring; width).\u003c/p\u003e\u003cp\u003eTwo-dimensional ROESY spectra recorded at concentrations favoring either the double helix (0.5 mM, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea) or the triple helix (5 mM, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) further supported our structural assignment. The two helices could be distinguished from the relative spatial arrangement of phenylene and pyridinium residues. For example, phenylene proton \u003cem\u003en\u003c/em\u003e showed through-space correlations with pyridinium proton \u003cem\u003ec\u003c/em\u003e in the double helix, but with pyridinium protons \u003cem\u003ef\u003c/em\u003e and \u003cem\u003eg\u003c/em\u003e in the triple helix. Likewise, phenylene proton \u003cem\u003ej\u003c/em\u003e correlated with pyridinium proton \u003cem\u003ef\u003c/em\u003e in the double helix, but with pyridinium proton \u003cem\u003ed\u003c/em\u003e in the triple helix. Additional diagnostic correlations are presented in Supplementary Figs. S27 and S28.\u003c/p\u003e\u003cp\u003eThese results further validate the predictive power of our sequence-to-structure model and demonstrate that bolaamphiphilic strands can be programmed to form multiple helical architectures in reversible exchange.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eControlling the conformational switch between double and triple helices: role of concentration, temperature, and anions.\u003c/b\u003e Having established that strand \u003cb\u003e2\u0026middot;OTf\u003c/b\u003e assembles into double and triple helices in dynamic equilibrium, we examined how external parameters influenced this conformational switch. Specifically, we investigated the effects of concentration, temperature, and anionic guests on the relative populations of the two helical states.\u003c/p\u003e\u003cp\u003eIn general, triple helix (\u003cb\u003e2\u003c/b\u003e)\u003csub\u003e3\u003c/sub\u003e was favored at higher concentrations and lower temperatures, while double helix (\u003cb\u003e2\u003c/b\u003e)\u003csub\u003e2\u003c/sub\u003e predominated under dilute conditions and elevated temperatures. Varying both parameters thus provided a reliable means to control the composition of the system. For instance, at 10 mM, the triple helix remained stable across a broad temperature range (288\u0026ndash;328 K, Supplementary Fig. S23). In contrast, at 3 mM, increasing the temperature from 288 K to 328 K induced a clear transition from the triple to the double helix (Supplementary Fig. S24).\u003c/p\u003e\u003cp\u003eWe next investigated whether the double-to-triple helix transition could be influenced by guest binding. As a starting point for this study, we compared the behaviors of strands \u003cb\u003e2\u0026middot;TFA\u003c/b\u003e and \u003cb\u003e2\u0026middot;OTf\u003c/b\u003e, which differ only in their counterions. The \u0026sup1;H NMR spectrum of \u003cb\u003e2\u0026middot;TFA\u003c/b\u003e at 5 mM in D₂O (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec) displayed upfield-shifted resonances characteristic of the triple helix, but these signals were significantly broader than those previously observed for \u003cb\u003e2\u0026middot;OTf\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). This difference suggested that the more chaotropic triflate occupies, at least partially, the cavity of the triple helix, thereby restricting its conformational motion. This interpretation was supported by the observation that adding potassium triflate \u003cb\u003eG1\u003c/b\u003e to a 5 mM aqueous solution of \u003cb\u003e2\u0026middot;TFA\u003c/b\u003e led to progressive sharpening and shifting of the triple helix resonances (Supplementary Fig. S30). The shifts were more pronounced for inner phenylene protons, as expected if triflate binds within the cavity. Fitting the titration data using a 1:1 binding model yielded an association constant of \u003cem\u003eK\u003c/em\u003eₐ = (9.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7) x 10\u003csup\u003e2\u003c/sup\u003e M⁻\u0026sup1;, approximately one order of magnitude higher than that previously measured for (\u003cb\u003e1\u003c/b\u003e)\u003csub\u003e2\u003c/sub\u003e. This increased affinity is attributed to the longer and more hydrophobic internal cavity of the triple helix (\u003cb\u003e2\u003c/b\u003e)₃, which provides a more favorable environment for guest encapsulation.\u003c/p\u003e\u003cp\u003eSimilar signal sharpening was observed upon addition of potassium perfluorobutanesulfonate (\u003cb\u003eG2\u003c/b\u003e, Supplementary Fig. S31) and sodium 4,4\u0026rsquo;-biphenyldisulfonate (\u003cb\u003eG4\u003c/b\u003e, Supplementary Figs. S32 and S33), indicating the formation of inclusion complexes (\u003cb\u003e2\u003c/b\u003e)₃\u0026sup;\u003cb\u003eG2\u003c/b\u003e and (\u003cb\u003e2\u003c/b\u003e)₃\u0026sup;\u003cb\u003eG4\u003c/b\u003e, respectively. Although the broad resonances at intermediate titration points precluded accurate determination of association constants for both guests, the importance of hydrophobicity in guest binding was highlighted once again by the lack of measurable interaction between sodium butanesulfonate \u003cb\u003eG3\u003c/b\u003e and the triple helix (Supplementary Fig. S29).\u003c/p\u003e\u003cp\u003eConcentration- and temperature-dependent NMR spectra recorded after addition of \u003cb\u003eG1\u003c/b\u003e, \u003cb\u003eG2\u003c/b\u003e and \u003cb\u003eG4\u003c/b\u003e (Supplementary Figs. S29-S39) demonstrated a clean transition between the double and triple helical states in the presence of these guests. A representative example of these experiments, obtained after addition of \u003cb\u003eG2\u003c/b\u003e, is shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-e. Comparisons between the effects of different anions on the switching behavior should be made with caution, as they depend on both the association constants with the double and triple helix, and the number of guest equivalents added. Nevertheless, the data suggest that more hydrophobic guests, expected to bind more strongly the triple helix, stabilize it and delay its transition to the double helix both upon dilution and heating. The most stable complexes, (\u003cb\u003e2\u003c/b\u003e)₃\u0026sup;\u003cb\u003eG2\u003c/b\u003e and (\u003cb\u003e2\u003c/b\u003e)₃\u0026sup;\u003cb\u003eG4\u003c/b\u003e, were modeled by DFT at the BP86-D4-COSMO(water)/def2-TZVP level of theory (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef-g), confirming a good geometric fit of these guests within the cavity of the triple helix. In the case of \u003cb\u003eG4\u003c/b\u003e, p-p stacking and electrostatic attraction between the aryl sulfonate moieties and the pyridinium units induces dynamic distortions of the triple helix, consistent with the slightly broader NMR signals obtained for the corresponding complex.\u003c/p\u003e\u003cp\u003eTaken together, these results demonstrate that three external parameters (concentration, temperature, and the presence of suitable anionic guests) can be used to reversibly control the conformational equilibrium between double and triple helices. Importantly, the switching process remained tightly controlled and cleanly operable under all tested conditions, with no detectable formation of the undesired single helices or strand unfolding.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTwo bolaamphiphilic oligo(\u003cem\u003em\u003c/em\u003e-phenylene ethynylene) strands were synthesized to demonstrate that the relative lengths of hydrophobic and charged segments dictate the formation of distinct helical architectures. As predicted, strand \u003cb\u003e1\u003c/b\u003e assembled exclusively into a stable double helix, whereas strand \u003cb\u003e2\u003c/b\u003e formed both double and triple helices in dynamic equilibrium. The latter system enabled clean, reversible switching between the permitted helical states in response to concentration, temperature, or binding of specific amphiphilic guests.\u003c/p\u003e\u003cp\u003eThis study establishes a robust strategy for controlling both the assembly and dynamic behavior of multistranded helices through the primary sequence of synthetic strands. A key strength of this approach lies in its minimalism: precise structural control was achieved using only two building blocks, hydrophobic phenylene and charged pyridinium residues. This simplicity renders the approach modular and potentially extensible to alternative sequence patterns (including non-bolaamphiphilic ones) and other aromatic foldamer scaffolds. Moreover, the oligo(\u003cem\u003em\u003c/em\u003e-phenylene ethynylene) backbone is amenable to chemical modification,\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e allowing for the integration of catalytic, recognition, or sensing functions within structurally programmed helices.\u003c/p\u003e\u003cp\u003eMore broadly, the ability to encode multiple, switchable helical states within a single strand introduces a new level of control in the design of adaptive molecular systems. By establishing a clear and predictable sequence-to-structure relationship, this work may enable the construction of complex, programmable supramolecular architectures with responsive functionalities.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available in the manuscript, in the Supplementary Information, or from the lead contact.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Research Council of Finland (grant 357271) and the University of Jyv\u0026auml;skyl\u0026auml;. A.F. and R.M.G. are grateful for project PID2023-148453NB-I00 funded by the Ministerio de Ciencia, Innovaci\u0026oacute;n y Universidades of Spain MCIU/AEI/10.13039/501100011033 and FEDER, UE.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.D., R.A. and P.V. performed chemical synthesis. D.D. and P.P. performed NMR analysis. A.F. and R.M.G. performed computational work. F.B.L.C. conceptualized and supervised the project. The paper was written through contributions from all authors, and all authors have given approval to the final version of the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary Information contains synthetic procedures, characterization data and theoretical calculations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNelson DL, Cox MM (2021) Lehninger Principles of Biochemistry, 8th edn. W. H. Freeman, New York\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBochman ML, Paeschke K, Zakian VA (2012) DNA secondary structures: stability and function of G-quadruplex structures. Nat Rev Genet 13:770\u0026ndash;780\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKaushik M et al (2016) A bouquet of DNA structures: Emerging diversity. 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Tetrahedron 60:10029\u0026ndash;10038\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7441733/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7441733/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSequence-specific conformational changes underpin essential biological processes, from information storage to energy transduction, but are difficult to replicate in synthetic systems. Here, we present a simple approach to encode in the primary sequence of molecular strands all the information required to govern both the formation and dynamic behavior of multistranded helices. We demonstrate that the sequence of oligo(\u003cem\u003em\u003c/em\u003e-phenylene ethynylene) strands composed of hydrophobic phenylene and charged pyridinium residues reliably direct the formation of either static structures (e.g., a double helix) or dynamic assemblies (e.g., double and triple helices in exchange). In the latter case, transitions between different helical states can be controlled by concentration, temperature, or by the presence of anionic molecules. This minimal yet versatile design strategy lays the groundwork for the construction of adaptive supramolecular systems with programmable structure and function.\u003c/p\u003e","manuscriptTitle":"Programmable Assembly of Multistranded Helices in Water","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-09 11:11:20","doi":"10.21203/rs.3.rs-7441733/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"656392ae-fe2a-49d3-aacc-83cec11ae1d7","owner":[],"postedDate":"September 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":54057719,"name":"Physical sciences/Chemistry/Supramolecular chemistry/Self-assembly"},{"id":54057720,"name":"Physical sciences/Chemistry/Organic chemistry/Structure elucidation"}],"tags":[],"updatedAt":"2025-12-12T08:09:41+00:00","versionOfRecord":{"articleIdentity":"rs-7441733","link":"https://doi.org/10.1038/s41467-025-67227-0","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-12-11 05:00:00","publishedOnDateReadable":"December 11th, 2025"},"versionCreatedAt":"2025-09-09 11:11:20","video":"","vorDoi":"10.1038/s41467-025-67227-0","vorDoiUrl":"https://doi.org/10.1038/s41467-025-67227-0","workflowStages":[]},"version":"v1","identity":"rs-7441733","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7441733","identity":"rs-7441733","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}