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Light-emitting self-assembled peptide nucleicacids exhibit both stacking interactions andWatson–Crick base pairingOr Berger1, Lihi Adler-Abramovich1, Michal Levy-Sakin2, Assaf Grunwald2, Yael Liebes-Peer2,Mor Bachar1, Ludmila Buzhansky1, Estelle Mossou3,4, V. Trevor Forsyth3,4, Tal Schwartz2,Yuval Ebenstein2, Felix Frolow1,5†, Linda J. W. Shimon6, Fernando Patolsky2,7 and Ehud Gazit1,7*

The two main branches of bionanotechnology involve the self-assembly of either peptides or DNA. Peptide scaffolds offerchemical versatility, architectural flexibility and structural complexity, but they lack the precise base pairing and molecularrecognition available with nucleic acid assemblies. Here, inspired by the ability of aromatic dipeptides to form orderednanostructures with unique physical properties, we explore the assembly of peptide nucleic acids (PNAs), which are shortDNA mimics that have an amide backbone. All 16 combinations of the very short di-PNA building blocks were synthesizedand assayed for their ability to self-associate. Only three guanine-containing di-PNAs—CG, GC and GG—could formordered assemblies, as observed by electron microscopy, and these di-PNAs efficiently assembled into discretearchitectures within a few minutes. The X-ray crystal structure of the GC di-PNA showed the occurrence of both stackinginteractions and Watson–Crick base pairing. The assemblies were also found to exhibit optical properties includingvoltage-dependent electroluminescence and wide-range excitation-dependent fluorescence in the visible region.

Nature has produced a basic set of building-block moleculesthrough billions of years of molecular selection and evolution.The 20 coded amino acids and four primary nucleotides are

the most fundamental elements in living systems. Protein andnucleic acid biomolecules allow the formation of an enormouslydiverse range of supramolecular assemblies, exhibiting a vast arrayof physical properties.

Inspired by natural ordered assemblies, many studies have beendirected towards the design of peptide and protein building blocksthat self-assemble into preferred architectures1–7. The shortest, mostsimple peptide building block shown to self-assemble into orderedarchitectures is diphenylalanine, the core recognition motif ofβ-amyloid polypeptide8. This aromatic dipeptide self-assembles intodiscrete well-ordered nanotubular structures of notable persistencelength in aqueous solution9. Various studies have shown the remark-able physical characteristics of these nanotubes, including metal-likerigidity and high thermal and chemical stability, as well as optical,semiconductive and piezoelectric properties10–12.

In contrast to peptide self-assembly, structural DNA nanotech-nology is solely derived from the specificity of the hydrogen-bonding interactions between complementary Watson–Crick basepairs. The use of DNA as a structural building block instead ofmerely a genetic material was first recognized in the early 1980s,in a theoretical work by Seeman13. Based on the complementarynature of nucleic acids, it is possible to predict and design DNAstructures of nanoscale order. This pioneering conceptual workmaterialized into a vivid field of research with tremendousgrowth. Now, with the aid of computer design and the DNA

origamimethod, the fabrication of any two- or even three-dimensionalnanostructure shape has become considerably simpler14–16.

Peptide and DNA building blocks offer two distinct approachesfor the generation of supramolecular architectures by self-assembly.Peptide-driven materials are characterized by robustness, syntheticversatility, architectural flexibility and structural complexity,whereas DNA nanostructures are based on specific molecular recog-nition and base-pairing complementarity. Convergence of thepeptide and nucleic acid assembly strategies could be very usefulfor the design of novel self-organized materials.

Peptide nucleic acids are artificially synthesized polymers that werefirst described by Nielsen and co-workers in 1991 (refs 17, 18). Thepolymer is an oligonucleotide analogue in which the phosphodiesterbackbone is replaced by repeating N-(2-aminoethyl) glycine unitslinked by amide bonds. Thus, PNA can be regarded as either aDNA mimic with a neutral amide backbone, or as a peptide mimicwith nucleobases as side chains. Another advantage of PNAs istheir resistance to degradation by proteases or nucleases. A slightlydifferent approach for the production of a peptide–nucleic acidhybrid is based on the conjugation of functionalized purine and pyr-imidine bases to a peptide backbone19. The neutral peptide-like back-bone replacing the negatively charged phosphodiester groups, whichmay limit self-association due to the repulsion of similar charges,adds structural elasticity and chemical adaptability. Another groupof unnatural peptide–nucleobases hybrids (denoted nucleopeptides)have already been shown to self-assemble into nanofibres to generatesupramolecular hydrogels20. Despite the great potential to convergethe two distinct fields of peptide self-assembly and structural DNA

1Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel. 2Schoolof Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Ramat Aviv 69978, Israel. 3Partnership for Structural Biology,Institut Laue Langevin, 71 Avenue des Martyrs, Grenoble Cedex 9 38042, France. 4Faculty of Natural Sciences, Keele University, Staffordshire ST5 5BG, UK.5Daniella Rich Institute for Structural Biology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel. 6Department ofChemical Research Support, Weizmann Institute of Science, Rehovot 76100, Israel. 7Department of Materials Science and Engineering, Iby and AladarFleischman Faculty of Engineering, Tel Aviv University, Ramat Aviv 69978, Israel. †Deceased. *e-mail: [email protected]

ARTICLESPUBLISHED ONLINE: 16 MARCH 2015 | DOI: 10.1038/NNANO.2015.27

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nanotechnology, no work has been performed on PNAs as buildingblocks to assemble into distinct structural entities by themselves. Asdescribed in a recent review21, PNA has so far only been used as atemplate or as a conjugate to other molecules that undergo the self-assembly process, in order to gain the hybridization properties ofnucleic acids. A notable example is the work of Stupp and co-workers22, in which a PNA strand and a peptide sequence that pro-motes nanofibre formation were coupled to form a PNA–peptideamphiphile conjugate. The designed fibre-shaped nanostructuresshow binding of oligonucleotides with high affinity and specificity.Here, we report for the first time the efficient and rapid formationof supramolecular architectures based solely on PNA moleculeself-assembly.

di-PNA synthesis, assembly and structureInspired by the ability of simple aromatic dipeptides to form uniqueassemblies, we decided to examine the ability of short di-PNA

building blocks to form ordered supramolecular assemblies. All ofthe 16 different di-PNA combinations were synthesized usingsolid-phase peptide synthesis (AA, AC, AG, AT, CA, CC, CG, CT,GA, GC, GG, GT, TA, TC, TG, TT; italics are used to denote PNA)(Fig. 1a). The synthesis of the di-PNA was performed according toconventional peptide synthesis practice using standard methodsand commercially available protected building blocks. The syntheticeffort, yield and number of steps are comparable to those forthe production of other aromatic dipeptides that are readilysynthesized at an industrial scale, such as L-aspartyl-L-phenylalaninemethyl ester.

Favourable conditions for self-organization were determined byscreening the di-PNAs in a variety of solvents including organicsolvents (methanol, ethanol, dimethyl sulphoxide, hexafluoro-2-propanol, and so on) and diverse buffer solutions with a range ofpH values and concentrations. Under alkaline conditions, micro-scopic observation of molecular assembly was evident with only

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Figure 1 | Self-assembly of guanine-containing di-PNAs into well-ordered architectures. a, The 16 different di-PNA building blocks were synthesized andassayed for their ability to undergo self-assembly into distinct structural entities. Molecular assembly was evident with three di-PNAs under alkalineconditions (highlighted in black). More assemblies were observed for three other di-PNAs on drying the sample (highlighted in grey). b–d, Chemicalstructures of the three assembly-forming di-PNAs: CG (b), GC (c) and GG (d). e–g, SEM micrographs of the structures formed by CG (e), GC (f) and GG (g).Scale bars, 10 µm. h, Light microscopy images of assemblies formed by GC di-PNA on dissolving with rising pH levels of disodium hydrogenphosphate buffer. Original magnification, ×40.

ARTICLES NATURE NANOTECHNOLOGY DOI: 10.1038/NNANO.2015.27

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three di-PNAs: CG, GC and GG (for structures see Fig. 1b–d).Examination of the solutions using light and electron microscopyrevealed well-organized architectures, including long rods (tens ofmicrometres long) for CG and GC (Fig. 1e,f ) and spheroids witha diameter of 2–3 μm for GG (Fig. 1g). Other di-PNAs did notform any type of ordered structure, or did so only upon drying ofthe solution (AG, GA and GT; Supplementary Fig. 1a–f ). Co-assem-bly of all the possible complementary di-PNA combinations at a50:50 molar ratio was also examined. Well-defined structures wereobserved solely for the combination of CC and GG. When mixedtogether, instead of the spherical assemblies formed by GG alone,elongated structures similar to those generated by CG or GC wereevident (Supplementary Fig. 1g). Based on these findings, we specu-late that it is necessary to have a minimum of six hydrogen bondsbetween the bases for the stabilization of complementary di-PNAsand for further organization into supramolecular entities.Interestingly, all PNAs that were able to form structures contained

the guanine nucleobase. As it is known that the secondary structureof G-containing PNA oligomers may be altered under alkaline con-ditions due to deprotonation of the guanine bases23,24, the assemblyof the GC di-PNAwas further examined under increasing pH levels(Fig. 1h). Furthermore, alkaline conditions have been found to beessential for self-assembly, presumably due to the introduction ofcharges, so high salt conditions (which may shield these charges)were examined. Accordingly, GC di-PNAwas dissolved in assemblybuffer with 1 M sodium chloride. Under this condition, only smallnucleation sites were observed. Typical structures initiated theassembly process following a tenfold dilution of the salt(Supplementary Fig. 1h).

In this context it should also be noted that guanine is a key com-ponent in the assembly of various natural nucleic-acid structures.Nucleic-acid sequences that are rich in guanine are capable offorming G-quadruplexes, the main structural motif of the telomericDNA. Moreover, guanosine analogues are able to self-associate into

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Figure 2 | Crystal structure of GC di-PNA. The GC di-PNA building block was crystallized and its structure determined by synchrotron radiation at aresolution of 0.95 Å. a, Molecular structure of a single GC di-PNA molecule from single-crystal structure determination. The cytosine and guaninenucleobases form an intramolecular stacking interaction. b, Each molecule forms hydrogen bonds with a neighbouring unit between the cytosine and guanineresidues. c, The hydrogen bond length between symmetry-related molecules is measured to be 2.85–2.93 Å, the same as in typical Watson–Crick base pairs.d, The bases are 3.5 Å apart, the same as in a DNA double-helix structure. e, The di-PNAs are packed in a continuous tilted stack through the crystal.f, When lining the stacks in the z direction it is evident that this form of packing results in rectangular-shaped pores comprising over 50% of thecrystal volume.

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dimers, ribbons and macrocycles that can further stack into supra-molecular assemblies25. As a control we studied the ability ofguanine-containing DNA dinucleotides to form ordered assemblies.No conditions under which ordered dinucleotides structures couldbe formed were found.

To gain better insight into the assembly process and stabilizinginteractions, we attempted to form ordered single crystals of theassemblies and acquire X-ray diffraction data. To this end, thedi-PNAs were screened for crystallization conditions. Crystals ofGC di-PNA were found to grow in a bicine-based crystallizationbuffer. Because bicine buffer also enables assembly of the structures,we strongly believe the crystal structure reflects the solution self-assembled architecture. The crystal structure of GC was determinedat a resolution of 0.95 Å with data collected at the EuropeanSynchrotron Radiation Facility (ESRF).

The determined structure revealed very unique packing of thePNA crystals. The cytosine and guanine in each molecule formstacking interactions (Fig. 2a). Each molecule then forms hydrogenbonds with a neighbouring unit between the cytosine and guanineresidues (Fig. 2b). All of the bases are engaged in Watson–Crickhydrogen bonds, as observed in canonical DNA–DNA andPNA–DNA duplexes26. The hydrogen bond length between

symmetry-related molecules was measured to be 2.85–2.93 Å(Fig. 2c), which is similar to that of Watson–Crick base pairing.The bases are found to be ∼3.5 Å apart (Fig. 2d), which is charac-teristic of DNA double-helical structures27, but they do not exhibitany tilt or roll. The hydrogen-bonding molecules are related toeach other via the two-fold dyad that passes between the stackedbases. Packing of the molecule in a centrosymmetric space groupis possible due to the non-chiral nature of the polyglycine backbone.The bases are packed in a continuous tilted stack throughout thecrystal (Fig. 2e). This form of packing results in rectangular-shaped pores that comprise over 50% of the crystal volume(Fig. 2f). The crystal structure reflects the dual identity of PNA.The di-PNAs form stacking interactions with each other, in thesame way as aromatic peptides, while at the same time formingthe Watson–Crick base pairs typical of DNA structures. Thisunique duality distinguishes these molecules from simple DNAdinucleotides that possess only Watson–Crick base pairing and donot form any self-assembled structures. It is also possible that thenegatively charged phosphate backbone of simple DNA oligomersmay limit the assembly properties.

The obtained crystal structure enabled us to estimate the rate atwhich the di-PNA monomers self-assemble in solution to form theordered structures. In Supplementary Movie 1 we capture theassembly process of the GC di-PNA in real time. Briefly, a thinglass capillary was filled with a fresh solution of the PNA buildingblocks and sealed immediately from both sides with wax toprevent evaporation and concentration changes of the solution.The capillary was monitored using light microscopy, and imageswere captured at a rate of one frame per second. Small nucleationseeds could be observed within a few seconds, and continualgrowth in one axis direction was sustained for a few minutes.Figure 3a depicts one capillary at five consecutive time points. Toallow better visualization of the elongation of the structure as a func-tion of time, one structure was tracked in a time frame in which theassembled structure is clearly seen and is not overlapped by otherarchitectures (Supplementary Fig. 2). Distinct elongating structuresfrom five different capillaries, each filled with freshly dissolved sol-ution, were examined for their growth rate, and an average of0.25 µm s−1 was measured. All the structures exhibited an excellentlinear fit between the dimension of the assemblies in the long Z-axisand time (R2 > 0.97). The elongation of a representative single struc-ture as a function of time is given in Fig. 3b. For this specific struc-ture, which is 1.77 µm wide, the elongation rate of 0.24 µm s−1

translates into a volume increase of ∼0.6 µm3 s−1. Because thecrystal unit cell has a volume of 11,676 Å3 (SupplementaryTable 1) and contains eight molecules, we can estimate that4.11 × 108 di-PNA building-block molecules organize into theordered structures each second. In comparison to other rapidelongating systems of a natural origin, such as the microtubulethat elongates by an average rate of 0.66 µm per minute28, the assem-bly kinetics of the di-PNA is over 20 times faster. When the sameexperiment was carried out in an open environment, instead of asealed capillary, the elongation rate was about ten times faster dueto evaporation of the solution, leading to higher local concentrationsof PNA. The rapid assembly of the di-PNA building blockssuggests they could be used in motor systems by convertingthe free energy emitted by the self-assembly process into mechanicalmotion29. Another very interesting property of this self-organiz-ation, important for technological applications, is its efficient,high-yield and uniform process, as only ordered homogeneousassemblies of the GC di-PNA could be observed under theexamined conditions.

Optical characterization of PNA assembliesWhen trying to examine whether the PNA structures could bindDNA intercalators due to their Watson–Crick base pairing, we

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Figure 4 | The PNA assemblies exhibit a red edge excitation shift with a broad range of emission wavelengths in the visible region. a, One bright-field andfive fluorescence images of the same microscopic field of GC di-PNA structures. Each fluorescence image was taken with different excitation and emissionfilters: (excitation/emission) 387 nm/440 nm; 485 nm/525 nm; 537 nm/578 nm; 560 nm/607 nm; 650 nm/684 nm (from left to right). Pseudo-coloursrepresent corresponding emission colour. Original magnification, ×100. b, Emission spectra of GC di-PNA assemblies at excitation wavelengths of 330, 340,350, 360, 370, 380, 390, 400, 410, 420 and 430 nm. The emission peak shifts to the red with higher excitation wavelengths. c, Graphical representation ofthe relation between the excitation and emission wavelengths. The slope is measured to be ∼0.7, which suggests a dynamic Stokes shift. d, Time-resolvedemission traces for different emission wavelengths following excitation at 390 nm. The emission onset is delayed with respect to the excitation peak asemission wavelength shifts to the red. e, Time delay of fluorescence as a function of wavelength. Δt is calculated to the fastest emission onset collected at410 nm. The monotonic increase in Δt is a signature of REES. f, Continuous model of solvent relaxation. The I state refers to one of the intermediate statesbetween the initial excited state and the final solvent relaxed state, in which the solvent molecules are partially relaxed. ν0, νI and ν∞ represent thefrequencies corresponding to the initially excited (Franck–Condon), intermediate and completely relaxed states, respectively, while λC and λR denote thewavelength maxima associated with these states.






made a surprising observation, as the control sample with no addeddye had a fluorescence signal similar to that of the stained samples.Even more surprising was the fact that fluorescence emission wasevident for a wide range of excitation wavelengths (Fig. 4a). Clearemission peaks were observed between 420 nm and 490 nm withthe excitation wavelength ranging from 330 nm to 430 nm. Fair fluo-rescent images could be obtained even for higher wavelengths, butwith lower intensity. As presented in Fig. 4a, the upper limit of theemission was observed at 684 nm when the excitation wavelengthwas 650 nm. To quantitatively analyse this phenomenon, fluor-escence emission spectra were determined at different excitationwavelengths (Fig. 4b). It is clearly noticeable from the studies thatthe emission peak shifts in the direction of the change in excitation.To determine whether the shift is consistent for various excitationwavelengths, we plotted consecutive emission maxima as a functionof excitation wavelengths (Fig. 4c). We observed a linear correlationbetween the excitation and emission peak wavelengths. Yet, the0.7073 slope of the very strong fit (R2 = 0.9943) indicates dynamicStokes shift behaviour as the distance between the excitation andemission peaks gradually decreases with longer wavelengths.

Such an observed change in fluorescence emission spectra inresponse to a shift in the excitation wavelength toward the rededge of the absorption band is termed a red edge excitation shift(REES). The phenomenon was originally described in rigid andhighly viscous environments such as low-temperature glasses, gra-phene oxide layers or highly condensed polymeric states30. REESis assumed to be the result of the strong reduction in the dynamicenvironment of the excited fluorophores in organized molecular set-tings. A model for this spectroscopic behaviour assumes that themolecular lattice confinements slow the rates of matrix relaxationand reorientation around the excited state of the fluorophore relativeto the fluorescence lifetime31. In biological and other organic mol-ecules, such constraints could be imposed by exceptionallyordered hydration shells or rigid membranes.

Time-dependent fluorescence measurements were used to deter-mine whether this model could explain the origin of the excitation-dependent emission behaviour observed with the PNA assemblies.The fluorescence decay was measured at several emission wave-lengths following excitation at 390 nm (Fig. 4d). The resultsclearly indicate a process of time-dependent fluorescence onset,with longer wavelengths appearing with larger delays after the exci-tation pulse (Fig. 4e) (for example, the emission at 510 nm appear-ing 700 ps after the emission at 410 nm). The optical phenomenonobserved here appears to be similar to the very strong REES effectshown in rigid graphene oxide31. The model used for the bulkcovalent carbon system may be applicable to the non-covalentPNA supramolecular system in that the dipole of the environmentmolecules (either solvent or PNA backbone) aligns gradually withthe excited PNA to minimize the interaction energy. Thus,various intermediate states are formed between the initial excitedstate and the final relaxed one (Fig. 4f ), resulting in the time-depen-dent fluorescence spectra collected and the observation of the shiftin the fluorescence emission maximum. Such optical behaviour wasnot observed in parallel peptide assemblies that lack the distinctstabilization with both stacking and base pairing, as observed inthe PNA crystal structure (Fig. 2). This indeed most likely representsa unique state of high polarizability in a motionally restrictedenvironment induced by the condensed lattice packing.

Intrigued by the optical properties distinguished for the charac-terized assemblies, we sought to study their capability to serve as anorganic light-emitting material in optoelectronics. A simple field-effect transistor (FET) device composed of a silicon chip withprinted gold source and drain electrodes was used (Fig. 5a). TheFET architecture enables the investigation of elementary optoelec-tronic properties in organic materials and is emerging as a highlyuseful configuration for applications such as optical communication

systems, advanced display technology, electrically pumped organiclasers and solid-state lighting32. The PNA structures were depositedin the gap between the source and drain electrodes on top of thechip. Using this simple platform, the electrical properties of theassemblies were measured. The measured resistance was found tobe between 0.1 and 1MΩ, and the conductance between 0.9 and1.8 μS. The PNA structures responded to gate voltage in the samemanner as a FET, with lower current rates as the voltage increased(Fig. 5b), and exhibited the nonlinear current–voltage characteristicof a diode (Fig. 5c). When applying different voltages to the device,electroluminescence was observed at both 5 V and −5 V. Figure 5dillustrates an experiment in which the voltage was alternated repeat-edly between 5 V and −5 V in time steps of 10 s. SupplementaryMovie 2 displays one minute of the experiment. As seen in bothFig. 5d and the movie, the device emits bright light every 10 s, asthe voltage reaches an absolute value of 5 V. The luminescencefades as the voltage is changed. The light produced by the deviceflickers dozens of times with no apparent decay.

The newly characterized assemblies should have potential foroptical biosensing and light emission-based applications includingorganic light-emitting diodes (OLEDs) and imaging labels withtunable emission via optical or electrical modulation. The blue lumi-nescence produced by the previously reported diphenylalaninenanostructures has raised considerable scientific and industrialinterest11. The novel PNA structures offer similar simplicity,

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prompt and efficient assembly, as well as the availability of industrystandard deposition methods such as physical vapour deposition,together with extended and tunable spectral properties and overan order of magnitude higher molar absorptivity relative to thediphenylalanine nanostructures.

ConclusionsWe have demonstrated the ability of PNA building blocks to self-assemble into ordered architectures. Very simple guanine-contain-ing di-PNA formed well-organized entities coordinated by bothstacking interaction as well as Watson–Crick base pairing. Theultrastructures show the combination of intramolecular organiz-ation with ordered supramolecular arrangement. The structureswere assembled into discrete and uniform entities, exhibiting veryfast elongation kinetics. The optical properties of the newly charac-terized assemblies are especially intriguing. The excitation-depen-dent emission and dynamic Stokes shift are unique for suchorganic supramolecular systems. Furthermore, the wide spectralspan may be useful for the fabrication of organic optical devices.This new and simple molecular system of notable physical proper-ties could serve as an excellent model for molecular self-assemblyas well as for the study of supramolecular polymers. The PNA archi-tectures could also serve in a variety of technological applications inthe fields of material science and bionanotechnology.

MethodsMaterials. Fmoc-protected PNA monomers were purchased from Polyorg, andFmoc-protected PAL-PEG-PS resin was purchased from Life Technologies. Allsolvents (peptide grade) used in the synthesis process were purchased fromBio-Lab. All crystallization solutions and equipment were purchased fromHampton Research.

PNA synthesis. di-PNAs were synthesized using standard solid-phase protocols24.The crude product was then purified by reversed-phase high-performance liquidchromatography using a C8 column. The product was verified by electrosprayionization time-of-flight mass spectrometry.

Scanning electron microscopy. Lyophilized PNA powder was dissolved in 0.1Mbicine buffer pH 9.0 to a concentration of 50 mgml−1. The solution was then dilutedwith ddH2O to a final concentration of 10 mgml−1. A 10 µl aliquot of the structuressolution was dried at room temperature on a microscope glass coverslip andcoated with chromium. Scanning electron microscopy images were taken using aJEOL JSM 6700F FE-SEM operating at 10 kV.

Crystallization and X-ray diffraction analysis. The di-PNAs were screened forcrystallization conditions using the hanging-drop vapour-diffusion method onsiliconized glass coverslips in Linbro plates, using 146 pre-formulated crystallizationsolutions. All crystallization experiments were performed at 293 K in a temperature-controlled room. After 5days, colourless needle-like crystals appeared for GC in0.1 M bicine pH 9.0, 2% vol/vol 1,4-dioxane, 10% wt/vol PEG 20,000.

Before mounting, crystals were soaked for 1 min in a cryo-protecting solution(comprising 16% ethylene glycol, 18% sucrose, 16% glycerol, 4% glucose, mixed ina 1:1 ratio with the crystallization reservoir solution). Crystals were mounted onloops and flash-frozen in liquid nitrogen for transportation to the synchrotron. Thedata were measured at ESRF beamline ID29 using a Pilatus 6M-F detector and awavelength of 0.80 Å. A full sphere of 360° of data were collected as 1° frames with aresolution of 0.95 Å (Supplementary Fig. 3). The data were autoprocessed usingEDNA33. Two data sets, collected from different locations on the same crystal, weremerged in XPREP. The structure was solved by direct methods in SHELXS. Therefinements in SHELXL-97 were weighted full-matrix least-squares against |F2|using all data. In the final stages of refinement, SQUEEZE34 was used due to the largevoids and remaining disordered solvent molecules. Atoms were refinedindependently and non-solvent atoms were refined anisotropically with theexception of hydrogen atoms, which were placed in calculated positions and refinedin a riding mode. Crystal data collection and refinement parameters are given inSupplementary Table 1 and the complete data can be found in the SupplementaryInformation (Cif file).

Spectroscopy measurements. Emission spectra were taken on a Horiba Jobin YvonFluorolog-3 spectrofluorometer at various excitation wavelengths as described inFig. 4b. Emission was recorded between 350 and 600 nm at 25 °C. Emission andexcitation slits were set at 2.5 nm. Measurements were performed in a 1 cmrectangular quartz cuvette containing 5 mgml−1 of GC di-PNA structures in buffersolution. All spectra were normalized so that the emission maxima and minimawere identical.

Time-resolved spectra were captured using time-correlated single photoncounting with a pulsed LED light source at 390 nm. The emission slit was set at10 nm. A stirred solution of 20 mgml−1 of GC di-PNA structures in buffer solutionwas used for the measurements.

Fluorescence imaging. A fresh solution of 5 mgml−1 of GC di-PNA structures wasprepared and a volume of 10 µl was deposited on a glass slide and covered with acoverslip. Images were acquired using five different excitation/emission filters asdescribed in Fig. 4a.

PNA-based electrical device array fabrication. The silicon wafers were cleaned bywashing with acetone, isopropyl alcohol, rinsing thoroughly with deionized water,blowing with dry nitrogen followed by oxygen plasma treatment (100 W, 50 s.c.c.m.O2 for 200 s; Axic HP-8). Source and drain electrodes were defined by maskexposure of a multilayer resist structure consisting of 500 nm LOR-5A copolymerand 500 nm S-1805 photoresist (MicroChem). After exposure, development andgold metallization of the gate, drain and source electrodes pattern (VST), the PNAassemblies, at a controlled density, were deposited between the source and drainelectrodes by the dropcasting approach. After the deposition step, the electrodeswere briefly washed with deionized water and dried gently with nitrogen. Theentire chip was then passivated with a 10–20 nm thin insulating dielectric layer ofSi3N4, deposited by inductively coupled plasma-enhanced chemical vapourdeposition (Axic).

Electrical characterization. I–V measurements were taken for each device using aprobe station (Janis Research) at room temperature. The drain current (Ids) responseto the applied Vds, varying between −5 V and +5 V at a rate of 100 mV s−1, wasrecorded at constant gate bias (taken from +6 V to −6 V).

Received 7 May 2014; accepted 2 February 2015;published online 16 March 2015

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AcknowledgementsThis work was supported in part by grants from the Israeli National NanotechnologyInitiative and Helmsley Charitable Trust for a focal technology area on Nanomedicine forPersonalized Theranostics. The authors thank members of the Gazit Laboratory for helpfuldiscussions, Y. Salitra for help with PNA synthesis andO. Yaniv for advice on crystallizationexperiments. The authors acknowledge the ESRF for synchrotron beam time and the staffscientists of the ID29 beamline for their assistance. O. Berger is supported by a fellowshipfrom the Argentinean Friends of Tel Aviv University Association.

Author contributionsO.B., L.A-A., L.B., Y.E. and E.G. designed the study. O.B., M.L-S., Y.L-P. and M.B.performed the experiments. O.B., A.G., T.S. and Y.E. analysed the data. F.F., L.J.W.S., E.M.and T.F. performed and analysed the X-ray diffraction experiments. F.P. performed OFETexperiments. O.B., L.A-A. and E.G. prepared the manuscript.

Additional informationSupplementary information is available in the online version of the paper. Reprints andpermissions information is available online at www.nature.com/reprints. Correspondence andrequests for materials should be addressed to E.G.

Competing financial interestsThe authors declare no competing financial interests.





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