The design and characterization of nucleo-peptides for hydrogel self

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The design and characterization of nucleo-peptides for hydrogel self-assembly Kiheon Baek, Alexander Noblett, Pengyu Ren, and Laura J. Suggs ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00229 • Publication Date (Web): 14 May 2019 Downloaded from http://pubs.acs.org on May 14, 2019

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Figure 1 Molecular structure and library of nucleo-tripeptides. 84x33mm (600 x 600 DPI)

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Table 1 Gelation conditions and properties of nucleo-tripeptide hydrogels. 1 wt% of Ade- and Thy- hydrogels and 1.5 wt% of Cyt- and Gua- hydrogels were used for measuring storage (G’) and loss (G’’) modulus. 172x96mm (300 x 300 DPI)


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Figure 2 Nano-fiber structures of self-assembled nucleo-tripeptide hydrogels were observed via transmission electron microscopy (TEM) images. (scale bar = 200 nm) 84x82mm (300 x 300 DPI)



Figure 3 Mechanical properties of Ade-FFF with concurrent strain profile demonstrated the self-healing ability of Ade-FFF (A). An Ade-FFF hydrogel injection through a needle was evidence of self-healing (B). 84x84mm (300 x 300 DPI)


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Figure 4 Circular dichroism spectrum of various concentrations of nucleo-triphenylalanine indicated peptide secondary helical structure and DNA-like stacking structure (A-D). Ramachandran plots of Ade-FFF and CytFFF models also supported the existence of peptides having phi and psi angles in a helical structure (E, F). 168x194mm (300 x 300 DPI)



Figure 5 MD simulation results of Ade-FFF and Cyt-FFF models supported their experimentally determined structure and properties. Stabilized SASA and RMSD of both models showed equilibrated assembled structures (A, B). Cyt-FFF had more hydrogen bonds between molecules and nucleobases (C), but a higher number of π-π stacking of nucleobases was seen in Ade- FFF model (D, E). Concurrently, the Ade-FFF hydrogel demonstrated a higher storage modulus (F). 169x211mm (300 x 300 DPI)


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Figure 6 SASA and RMSD of heterogeneous models (A, B). The number of hydrogen bonds between nucleobases of (Cyt-,Gua-) FFF was drastically increased by Watson-Crick interaction (C,E), and its frequency of nucleobases’ π-π stacking was similar with (Ade-, Thy-)FFF model (D). These combined effects showed more increased storage modulus when mixing Cyt-FFF and Gua-FFF for forming a hydrogel (F). 166x209mm (300 x 300 DPI)



Figure 7 The gelation conditions, specifically pH and concentration, of nucleo-tripeptides following gelation (A) and results from an in vitro biocompatibility study of nucleo-triphenylalanines (B). No significant differences were seen among any of the samples at any concentration and controls. 84x129mm (300 x 300 DPI)


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The design and characterization of nucleo-peptides for hydrogel self-assembly Kiheon Baek a, Alexander D. Noblett a, Pengyu Ren a, and Laura J. Suggs *a

a

Department of Biomedical Engineering, The University of Texas at Austin, Austin, Texas

78712, United States KEYWORDS: Self-assembly, nucleo-peptides, hydrogel, molecular dynamics simulation, supramolecular chemistry, nanofiber, biocompatibility

ABSTRACT

Self-assembling peptides can be used in a bottom-up approach to build hydrogels that are similar to the extracellular matrix at both structural and functional levels. In this study, a nucleotripeptide library was constructed to identify molecules that form hydrogels under physiological conditions. We used both experimental and computational approaches to study these selfassembled structures. Circular dichroism spectroscopy, transmission electron microscopy, and rheometry were utilized to support and supplement molecular dynamics simulations. Our data demonstrate that nucleo-tripeptides can form nano-fibrous hydrogels through Watson-Crick base pairing and π-π stacking interactions. Self-assembly conditions are mediated by nucleo-


1

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tripeptide hydrophobicity and amphiphilicity and can therefore be regulated by rational molecular design. We have found that structures derived from specific peptide and nucleobase conjugations form hydrogels under physiologic conditions, making them promising candidates for biomedical applications.

Introduction Peptide-based biomaterials have numerous biomedical applications due to their biocompatibility, potential for modification, and capacity to self-assemble into hydrogels structurally similar to the extracellular matrix (ECM). The 20 standard proteogenic amino acids have diverse molecular characteristics, and peptide materials are readily made using a bottom-up approach.1,2 The features of self-assembled peptides, which include their biological diversity, chemical versatility, and wealth of studies regarding structure and synthesis, have increased their popularity in biomaterials research. The combination of molecular secondary interactions and peptide secondary structures (e.g. α-helices, β-strands, β-turns, random coils, and coiled coils) gives a myriad of possibilities for controlling self-assembly and developing new structures.3 Further, peptide sequence can confer specific biologic functionalization to constructs, including targeting and degradation.4,5

Many self-assembling peptide molecules and peptide-based structures have been studied, including peptide amphiphiles, secondary structure peptides, and short modified peptides, leading to a variety of structures, such as micelles, vesicles, fibers, ribbons, and tubes.6-9 These materials are used in multiple applications, such as functional cell scaffolds for tissue


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engineering, targeted delivery carriers for controlled release, and templates for guiding nanostructure development. Our group has focused on hydrogels composed of short peptide derivatives in order to study self-assembly processes and develop tissue engineering scaffolds. Short modified peptides have several advantages in the study of self-assembly due to their ease of synthesis and controllable molecular interactions leading to association and supra-structure formation.10,11 Self-assembled peptide-based hydrogels are also well-suited for tissue engineering applications as a result of many ECM-like characteristics, particularly morphology, physical properties, and enzymatic susceptibility.12-16

Our group has reported on the self-assembly of depsipeptides, which have alternating ester and amide bonds along the polymer backbone. We demonstrated experimentally and computationally in short peptides modified by fluorenylmethyloxycarbonyl (Fmoc) that π-π stacking of aromatic groups and hydrophobic considerations are the primary driving forces for self-assembly, while β-sheet interactions were minimal.17 Based on this discovery, a selfassembled, hydrolytically degradable Fmoc-depsipeptide hydrogel was developed containing a cell-attachment peptide moiety, arginine-glycine-aspartic acid (RGD). The hydrogel degraded linearly over a period of 60 days and demonstrated increased cell attachment in vitro, but the viability of cells was reduced over time.18 We suggested that this decrease may be caused by chemical toxicity of the degradation products or by a reduction in physical properties during degradation.19-21

In the current study, we aimed to design a small self-assembling nucleo-peptide library. These molecules have a nucleobase, which we postulate may have reduced toxicity relative to


3

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Fmoc, at the N-terminus of the peptide chain. We also hypothesize that nucleobases provide an opportunity to enhance molecular self-assembly. Both of these features would be advantageous in the development of a biocompatible hydrogel that assembles in a physiological environment. In particular, we are interested in molecules that assemble at low concentrations and neutral pH in order to maintain high cell viability in culture. The candidate molecules were derived from a previously reported template of Nuc-FF (Nuc: nucleobase including Ade: adenine, Thy: thymine, Cyt: cytosine, Gua: guanine, F: phenylalanine), which form nano-fibrous structures at above 2 wt% and below physiological pH (~5).22-24

Experimental Section Materials All chemical compounds were purchased from Milipore Sigma (Milwaukee, WI), and Fmoc-Phe-Wang was purchased from Bachem (Torrance, CA).

Synthesis of nucleotide derivatives Nucleobase acetic acids, except thymine acetic acid, were prepared in 15 mmol scale based on the synthetic route (Fig S1) from Porcheddu et al. and Mercurio et al.25,26

Adenine or cytosine (1 equiv.) and 4-dimethylaminopyridine (DMAP, 0.1 equiv.) were dissolved in dry tetrahydrofuran (THF) and di-tert-butyl decarbonate (Boc O, 4 equiv.) was 2


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added to the reaction batch. The reaction mixture was stirred overnight at room temperature and excess THF was evaporated the following day. The product was dissolved in methanol (MeOH) and saturated sodium bicarbonate (NaHCO aq.) was added. The reaction mixture was stirred for 3

1 hour at 50°C, then water was added to the suspension after evaporating MeOH. The product was extracted with dichloromethane (DCM, 3 times) and the collected organic layer was dried with sodium sulfate (Na SO ), filtered, and evaporated to give a Boc-protected nucleobase (98%). 2

4

In order to attach a methyl acetate linker, Boc-protected nucleobase (adenine or cytosine) was dissolved in THF and sodium hydride (NaH, 1.2 equiv.) was added. The solution was placed in an ice bath to decrease the reaction temperature to 0 °C. Methyl bromoacetate (1 equiv.) was added dropwise, then the ice bath was removed after 1 hour to react overnight at room temperature. Water was added to quench the reaction and THF was evaporated. The residue was extracted with DCM (4 times) and the collected organic layer was dried with Na SO , filtered, 2

4

and evaporated. The product was then purified by flash chromatography on a silica gel column (Ethyl acetate:Hexane 5%-95% gradient) to obtain pure Boc-protected nucleobase with a methyl acetate linker (93%). To remove the methyl protecting group, the above compound was dissolved in H O/THF 2

(1:1) and cooled to 0 °C in an ice bath. Sodium hydroxide (NaOH, 1.2 equiv.) was added dropwise and the reaction mixture was stirred for 30 minutes. Hydrochloric acid (HCl, 1N) was added to decrease pH to 2-3 and the solution was extracted with ethyl acetate (4 times). The product was then dried with Na SO , filtered, and evaporated to give a Boc-protected nucleobase 2

4

acetic acid (82%).


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For guanine acetic acid synthesis, 2-amino-6-chloro purine was used as a starting material because guanine has very low solubility in organic solvents. The same Boc protection and methyl-protected linker attachment procedures were performed first. To replace chlorine at the purine ring with an oxo-group, the compound was dissolved in 1,4-dioxane and potassium acetate (CH COOK, 3 equiv.) and triethylamine (TEA, 3 equiv.) were added. 1,43

diazabicyclo[2.2.2]octane (DABCO, 0.1 equiv.) was then added portion-wise while stirring and the reaction batch was stirred overnight at room temperature. Saturated NaHCO solution was 3

added and after 30 minutes the solution was washed with ethyl acetate. The organic layer was back-extracted with saturated NaHCO solution (2 times). The combined aqueous layer was 3

placed in an ice bath and the pH was decreased to 2-3 by adding 1N HCl. The acidic aqueous phase was then extracted with ethyl acetate (4 times) and the collected organic layer was dried with Na SO , filtered, and evaporated to give the product (91%). 2

4

To remove the methyl protecting group at the acetate linker, the above compound was dissolved in THF and placed in an ice bath. Sodium hydroxide solution (2.2 equiv.) was added dropwise and the reaction proceeded for 1 hour at room temperature. The mixture was cooled to 0 °C and 1N HCl was added to decrease the pH to 2-3. Ethyl acetate extraction was performed, and the collected organic layer was washed with saturated sodium chloride (NaCl) solution, dried with Na SO , filtered, and concentrated to give the guanine acetic acid product (89%). 2

4

Solid phase synthesis of nucleo-tripeptides All nucleo-tripeptides were made (0.25 mmol scale) by traditional Fmoc solid phase peptide synthesis and coupled on solid phase using diisopropylcarbodiimide (DIC) / OxymaPure


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(ethyl 2-cyano-2-(hydroxyimino)acetate) amide coupling chemistry. Fmoc-Phe-Wang resin was swelled in DCM in a fritted, capped syringe for 20 minutes, then rinsed with dimethylformamide (DMF). Five milliliters of 20% piperidine in DMF was used for deprotecting the Fmoc group (20 minutes X 2 times). After washing with 5 mL of DMF (3 times) and 5 mL of DCM (2 times), the resin was mixed with a coupling solution (3 equiv. of Fmoc protected amino acids or 2 equiv. of nucleobase derivatives, 3 equiv. of OxymaPure, and 3 equiv. of DIC) for 50 minutes. The coupling reaction was followed by 4 times of 10 mL DMF washing and 2 times of 10 mL DCM. Fmoc deprotection and coupling were repeated until achieving the desired nucleo-peptide. Cleavage from the resin and side group deprotection were simultaneously completed by 2.5-3 hours reaction with 5 mL solution of 95:2.5:2.5 trifluoroacetic acid:water:triisopropylsilane (TFA:H O:TIPS). The mixture was then collected in a clean round bottom flask, and subsequent 2

resin washes (7 X 10 mL DCM) were added to the flask. After concentrating in vacuo on a rotary evaporator, the resulting viscous solution was precipitated with cold diethyl ether. Scavenged protecting groups were removed by 3x washing with cold diethyl ether and centrifugation, then the product was dried under a gentle stream of N . By re-dissolving in 1:1 water:acetonitrile, the 2

product was purified by reversed phase- (RP-) HPLC (5% H O initially for 5-10 minutes, 2

followed by a 25-30 minutes linear gradient of 5-95% acetonitrile in water). Pure product fractions were collected and lyophilized in a low temperature (-100 °C collector) for 2 days.

Gelation of self-assembling nucleo-peptides A pH switch method was employed for inducing self-assembly of nucleo-tripeptides. Briefly, each nucleo-peptide was dissolved in 200 μL H O and 40 μL of 0.5 M NaOH and gently 2


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mixed until clear. An adequate volume of 0.5 M HCl was slowly added and gently mixed until the assembled structure was formed in the solution. The inverted vial method was used to confirm hydrogel formation. In the case of mixture hydrogels, both nucleo-tripeptide powders were dissolved in 200 μL H O and 40 μL of 0.5 M NaOH, then 0.5 M HCl was gradually added 2

to induce self-assembly.

Gel rheology Storage and loss moduli of nucleo-peptide hydrogels or nucleo-peptide mixture hydrogels were measured by an Anton-Paar MCR101 rheometer with a parallel plate geometry (top plate diameter: 8 mm). Hydrogels were prepared by forming in 3 mm deep, 8 mm diameter polydimethylsiloxane (PDMS) molds. Oscillatory shear stress rheometry was performed (1% strain, 0.5-100 Hz).

Circular dichroism (CD) measurement Nucleo-tripeptide samples were pipetted into a cylindrical quartz cuvette with a 0.1 mm path length. Spectra between 185 nm and 400 nm were measured by a Jasco J-810 CD Spectrometer. The instrument was operated with a 0.5 nm data pitch, 200 nm/min scan speed, and 1 s response time. Each data point was the average value of three measurements.

Transmission Electron Microscopy (TEM) analysis


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FEI Tecnai TEM and negative staining with uranyl acetate were used for supra-structure analysis. The surface of a carbon-coated, 300-mesh copper specimen support grid (Electron Microscopy Services, Hatfield, PA) was initially ionized using a glow discharge system. Approximately 10 μL of each nucleo-peptide hydrogel was placed on the grid and was allowed to adsorb for five minutes. Excess sample was removed by using a Kimwipe and the grid was air-dried for five minutes. Two microliters of 2% uranyl acetate was placed on the dried grid for 5 minutes, then removed and dried before mounting in the TEM.

Molecular Dynamics (MD) simulations All simulations and calculations were performed using AMBER 1427 with AMBERff12SB force field28, with periodic boundary conditions and explicit water molecules. Non-standard residues, nucleobase acetic acid (ADL, THL, CYL, and GUL) and C-terminal protonated phenylalanine (AHP), were parameterized for building nucleo-tripeptide molecules. The charge and force field parameter of individual atoms in the residue were assigned by AM1BCC29 and Amber atom types respectively. AMBER GAFF force field30 was used to assign the missing parameters. Nuc-FFF molecules were built in AMBER with the parameterized nonstandard residues. Four peptide systems, Ade-FFF (120 molecules of Ade-FFF), (Ade-,Thy-)FFF (60 AdeFFF molecules and 60 Thy-FFF molecules), Cyt-FFF (Cyt-FFF 120 molecules), and (Cyt-,Gua)FFF (60 molecules of Cyt-FFF and 60 molecules of Gua-FFF), were selected for simulations.


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To build an initial simulation system, packmol31 was used for arranging 120 molecules randomly in the simulation box (100 Å cube), which was then solvated with TIP3P water molecules using the SOLVATEBOX in AMBER. Proper number of ions (Na+ and Cl-) were added for representing the ionic strength (0.1 M). Before the productive simulated annealing (SA) MD, energy minimization and equilibration simulations were applied. Initially, starting structures were subjected to two steps of energy minimization: minimization with solute restrain (500 cycles by steepest descent algorithm and 500 cycles by conjugate gradient algorithm) and full system minimization (1000 cycles by steepest descent algorithm and 1500 cycles by conjugate gradient algorithm), to remove high-energy contacts. Next, 100 ps NVT with solute restrained and 1 ns NPT simulations without restraints were performed to equilibrate the system. 50 ns SA MD simulations were then performed to simulate the assembly. The temperature of the system was increased to 500K during the initial 5 ns, and then gradually decreased to 300K in 45 ns followed by 5 ns equilibration at 300K. After SA MD, an additional 20 ns NPT equilibration was performed to analyze assembled structures. Hydrogen mass repartitioning was adopted in the NPT equilibration in order to use a time step to 3 fs.32 SHAKE algorithm was used to constrain covalent bonds with hydrogen atoms, allowing a 2 fs time step for MD simulations.33 Constant temperature and pressure were maintained by Langevin thermostat34 and Berendsen barostat35, respectively.

In vitro biocompatibility assay


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In order to evaluate the cytotoxicity of our synthesized nucleo-peptides, an MTS [3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, CellTiter 96, Promega] assay was performed to measure the proliferation 3T3 fibroblasts in culture with media containing various concentrations of four nucleo-peptides. Briefly, 104 cells/cm2 of 3T3 fibroblasts (after at least 2 passages) were seeded on 48-well microplates. After 24 hours of culture with complete media, the media was replaced by nucleo-peptide-containing media (0.6-0.075 wt%), and fibroblasts were incubated for 16 hours. Because the chosen concentrations were below their critical gelation concentration, they reduced the media pH more than in the gelled form. As a result, the pH of the media was neutralized by adding 0.1M NaOH prior to incubation with cells. The proliferation of fibroblasts after 16 hours incubation was measured by MTS according to the manufacturer’s protocol.

Results and Discussion Design and synthesis of nucleo-tripeptides Sixteen nucleo-tripeptides were designed based on the nucleo-diphenylalanine previously reported.24 (Figure 1) Nucleo-diphenylalanine molecules were shown to form nano-fibers leading to gelation, but the self-assembly conditions via pH change, namely high concentration (2 wt%) and low pH (~5) of the resulting hydrogel are not ideal for cell culture applications. In order to raise the pH of gelation, increase potential self-assembly interactions, and control the amphiphilicity, we decided to make a small library of nucleo-peptides. We inserted an additional amino acid between the nucleobase and diphenylalanine. Four amino acids having different side


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chain characters were chosen: bulky, hydrophobic phenylalanine (F), small, hydrophobic alanine (A), small, flexible glycine (G), and charged, hydrophilic lysine (K). Each nucleobase acetic acid (except thymine acetic acid) was produced by following a similar synthetic route (Fig S1), and these were used for solid-phase peptide synthesis of nucleo-tripeptides. Sixteen nucleotripeptides were successfully synthesized, purified, and lyophilized for further analysis.

Figure 1 Molecular structure and library of nucleo-tripeptides.

Self-assembly properties of nucleo-tripeptides In order to study the self-assembly of our nucleo-tripeptides, we developed a protocol for inducing gelation in our small molecules via pH change. All of 16 nucleo-tripeptides were well dissolved in basic aqueous solution. A solution of 0.5 M hydrochloric acid was gradually added to nucleo-tripeptide solutions leading to neutralization of the carboxylic acid group of the Cterminus and formation of the structure. The gelled supra-structures were investigated by the inverted-vial test. (Table 1) We found that all of the nucleo-tripeptides except Gua-KFF were able to form hydrogels. Next, TEM was used to analyze the self-assembled structure at the nanoscale. (Figure 2) All nucleo-tripeptide hydrogels with FFF and AFF formed nano-fibers in which the diameter was in the range 6~18 nm. Ade-GFF hydrogel also had network structure of nano-fibers, but other nucleo-tripeptides with the GFF sequence did not. In the case of Thy-GFF


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and Gua-GFF, micro-scale crystals were observed. Interestingly, Cyt-GFF was initially assembled into a nano-fibrous structure, but this structure was lost over time and changed to ~100 nm connected spherical aggregates. Nucleo-tripeptides having KFF generally showed larger nano-fibers (20 nm) than other hydrogels. Table 1 Gelation conditions and properties of nucleo-tripeptide hydrogels. 1 wt% of Ade- and Thy- hydrogels and 1.5 wt% of Cyt- and Gua- hydrogels were used for measuring storage (G’) and loss (G’’) modulus.


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Figure 2 Nano-fiber structures of self-assembled nucleo-tripeptide hydrogels were observed via transmission electron microscopy (TEM) images. (scale bar = 200 nm)

In comparing the formed hydrogels based on the hydrophobicity of the additional peptide insertion (-FFF, -AFF, -GFF), the more hydrophobic nucleo-tripeptides tended to form selfassembled structures at lower concentrations and higher pHs. This result confirms that hydrophobicity and π-π interactions are the primary driving force for assembly.36 Furthermore, a hydrophobic environment constructed by hydrophobic peptides increased the pKa of the Cterminal carboxylic acid group, thus raising the pH for gelation.37 On the other hand, nucleotripeptides having glycine between the nucleobase and diphenylalanine required a higher concentration and resulted in a lower pH for gelation. Except for Ade-GFF, other GFFcontaining molecules either formed crystals or, in the case of Gua-GFF, lost their gelled form rapidly, after 10 minutes. This more transient gelation was evidence of the negative effect of increased flexibility and the decreased molecular interaction conferred by glycine.38


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Interestingly, unlike the other peptide sequences, nucleo-tripeptides containing lysine formed gels at a similar pH to those with -FFF but took a longer time for gelation. (Both AdeKFF and Thy-KFF showed a change in optical transparency after approximately 20 minutes, and sonication was required to promote gelation for Thy-KFF.) Presumably, protonated lysine side chains lead to higher pH values, while the increased overall hydrophilicity of the molecule may limit the driving force for gelation and increase the time for self-assembly.

Mechanical properties of nucleo-tripeptide hydrogels Rheological characterization of nucleo-tripeptide hydrogels was performed in order to compare their mechanical properties. (Table 1) Higher storage than loss moduli of nucleotripeptide self-assembled structures demonstrated that elastic hydrogels were formed. The formed hydrogels achieved storage moduli on the order of 10Pa-1kPa. This modulus is appropriate for the engineering of soft tissues having a similar stiffness to endothelial tissue.39 Intriguingly, nucleo-tripeptide hydrogels having a -KFF sequence showed much higher storage and loss moduli than other relevant nucleo-tripeptides. These unusual mechanical properties may have derived from the charge interaction via lysine’s protonated amine group. One of the important features of this family of self-assembled hydrogels is the ability to self-heal, recovering its fiber network formed by secondary interactions. We evaluated this property by conducting a dynamic strain step test on the Ade-FFF hydrogel. (Figure 3) The storage modulus immediately dropped from approximately 1800 Pa to 10 Pa when subjected to a 100% strain, but both storage and loss modulus were immediately recovered when the strain


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returned to 0.1%. The recovered storage modulus maintained its initial storage modulus, indicating that Ade-FFF may restore the secondary interactions leading to hydrogel formation in spite of repeated strain stress. This self-healing ability makes the Ade-FFF hydrogel suitable as an injectable material which will be useful for biomedical applications.

Figure 3 Mechanical properties of Ade-FFF with concurrent strain profile demonstrated the self-healing ability of Ade-FFF (A). An Ade-FFF hydrogel injection through a needle was evidence of self-healing (B).

Molecular dynamics simulations of nucleo-tripeptide self-assembly We carried out MD simulations for detailed analysis of nucleo-triphenylalanines’ selfassembled structure. Simulated annealing was performed to predict the assembled structures from initial random configurations. Four peptides, Ade-FFF (120 molecules of Ade-FFF), (Ade,Thy-)FFF (60 Ade-FFF molecules and 60 Thy-FFF molecules), Cyt-FFF (120 molecules of CytFFF), and (Cyt-,Gua-)FFF (60 molecules of Cyt-FFF and 60 molecules of Gua-FFF), were


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selected for analyzing both the final self-assembled structures and the effect of nucleobase substitution. Each chosen model represented a purine ring, a pyrimidine ring, the adeninethymine pair, or the cytosine-guanine pair, respectively. The simulation results were also compared with experimental data in order to characterize the self-assembled nucleo-tripeptide structure from a molecular perspective. After 50 ns of simulated annealing and an additional 20 ns of equilibration, selfassembled structures were observed from all models, and the convergence of SASA and RMSD indicated the structural stability. We analyzed surface properties of assembled structures by using a linear combination of pairwise overlaps (LCPO) algorithm40 during equilibration, and hydrogen bonding within Watson-Crick interactions was searched according to distance (3Å between acceptor and donor heavy atom) and angle (>135° bond) conditions. Nucleobase π-π stacking was examined based on 3 conditions, minimum distance between two bases’ heavy atom (

The design and characterization of nucleo-peptides for hydrogel self

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