PHYSICAL REVIEW RESEARCH 4, 033046 (2022) Smectic-B phase and temperature-driven smectic-B to -A transition in concentrated solutions of “gapped” DNA Prabesh Gyawali ,1 Rony Saha,1 Sineth G. Kodikara,1 Ruipeng Li,2 Masafumi Fukuto,2 James T. Gleeson,1 Gregory P. Smith,3 Noel A. Clark,3 Antal Jakli ,1,* Hamza Balci,1 and Samuel Sprunt1,† 1 2 Department of Physics, Kent State University, Kent, Ohio 44242, USA National Synchrotron Light Source II, Brookhaven National Laboratory, Upton, New York 11973, USA 3 Department of Physics, University of Colorado, Boulder, Colorado 80309, USA (Received 22 February 2022; accepted 16 May 2022; published 18 July 2022) The occurrence of a smectic-B (Sm-B) phase is demonstrated in concentrated aqueous solutions of “gapped” DNA constructs consisting of fully paired duplexes bridged by a flexible, unpaired strand of nucleotides. The Sm-B phase, identified by small and wide angle x-ray scattering measurements and optical microscopy, develops from a smectic-A (Sm-A) phase with increasing DNA concentration at room temperature. It transitions (reversibly) to the Sm-A when the temperature is raised above ∼50 ◦ C. DOI: 10.1103/PhysRevResearch.4.033046 I. INTRODUCTION In recent years, studies on mesophasic self-assembly in aqueous solutions of ultrashort DNA duplexes or their oligomeric precursors [1–4], together with investigations of elementary lamellar (smectic-A [Sm-A]) ordering of “gapped” DNA (GDNA; duplexes bridged by a single strand [ss] of unpaired nucleotides) [5,6] and of fully paired duplexes capped at one end by a short hairpin [7], have reinvigorated research into the liquid crystalline (LC) properties of the molecule of life. Building on classic work on cholesteric and columnar LC phases in solutions of duplex DNA [8–12], the new developments point to richer mesomorphic behavior that is achievable by simple manipulations of the morphology of individual DNA constructs. The Sm-A phase of aqueously dispersed GDNA occurs in the concentration range cDNA ∼ 230−260 mg/ml, largely at the expense of the cholesteric state. It exhibits a significant sensitivity to temperature and, depending on the length of the gap, features either a mono- or bilayer structure (∼1 or 2 duplex layer spacing, respectively) [6]. The smectic ordering and its temperature dependence were attributed to a combination of enthalpic interactions and entropic effects: Hydrophobic attraction between blunt duplex ends favors end-to-end stacking of the duplex segments between GDNA constructs, while segregation of the flexible ss “gaps” into layers becomes entropically favorable when the constructs are densely packed. * Advanced Materials and Liquid Crystals Institute, Kent State University, Kent, OH 44242. † ssprunt@kent.edu; Advanced Materials and Liquid Crystals Institute, Kent State University, Kent, OH 44242. Published by the American Physical Society under the terms of the Creative Commons Attribution 4.0 International license. Further distribution of this work must maintain attribution to the author(s) and the published article’s title, journal citation, and DOI. 2643-1564/2022/4(3)/033046(7) 033046-1 The present work addresses the questions: What happens to the Sm-A phase in GDNA, and what role does temperature play at higher DNA density? When concentrated above the cholesteric state, fully paired duplexes transition to a 2D hexagonal columnar phase at cDNA ∼ 400 mg/ml and then, at ∼500−700 mg/ml, they form 3D hexagonal and orthorhombic crystals [11,12]. Here we demonstrate that LC solutions of GDNA constructs transform to a higher-order smectic phase, which we identify as a lyotropic smectic-B (Sm-B) phase, at substantially lower concentrations (260−290 mg/ml) and undergo a thermotropic transition to the Sm-A phase in the ∼55−60 ◦ C temperature range. Our results further expose the distinct and remarkably rich LC properties of DNA constructs that incorporate rigid and flexible elements. They are also compelling since Sm-B phases appear to be relatively rare in lyotropic systems; to our knowledge, prior reports are limited to colloidal suspensions of charged, inorganic platelet- [13] or rod- [14] shaped particles and dense solutions of semiflexible rodlike viruses [15–17]. II. EXPERIMENTAL DETAILS The investigated GDNA construct, denoted 48-10T-48, consists of two, symmetric 48 bp duplexes connected by a ss “gap” of 10 unpaired thymine bases (Fig. 1). The construct was synthesized from PAGE-purified oligomers purchased from ExonanoRNA (Columbus, OH). Details of the synthesis and the specific nucleotide sequences are described elsewhere [6]. For small/wide angle x-ray scattering (SAXS/WAXS) studies, we prepared dense aqueous solutions of 48-10T-48 constructs in standard borosilicate capillaries with a 1.25-mm inner diameter as follows: We first slowly and evenly loaded a ∼40 μl volume of isotropic solution (cDNA ∼ 80−100 mg/ml) into an open capillary and then very gradually allowed the water component of the buffer to evaporate until a target volume was reached. The components of the initial Published by the American Physical Society PRABESH GYAWALI et al. PHYSICAL REVIEW RESEARCH 4, 033046 (2022) FIG. 1. Top: Schematic structure of the 48-10T-48 GDNA construct studied. Middle: Optical texture of smectic FC “fan” domains in a thin film of 48-10T-48 GDNA solution sandwiched between glass slides at 25 ◦ C. The dashed yellow line separates distinct “smooth” and “striated” FC textures (to the left and right of the line, respectively) that are observed in the presence of a positive DNA concentration gradient running from left to right. Scale bar = 50 μm. Bottom: The black/white images on the left and right detail smooth and striated FC “fan backs” in two samples sealed at lower/higher GDNA concentration (cDNA ). The accompanying images in color were taken with a quartz compensator inserted in the optical path. Relative color shifts observed in radial directions from the “tip” of an isolated FC that are along and normal to the compensator’s optical axis (labeled e) confirm radial orientation of the GDNA duplexes. The textures are consistent with smectic layers that bend along the azimuthal direction in the FCs and with the formation of a higherorder smectic phase (striated texture) at higher cDNA . buffer solution were adjusted so that after the water evaporation, the buffer composition was approximately 150 mM NaCl/10 mM Tris/0.1 mM thylenediaminetetraacetic acid (EDTA) in deionized water at pH 7.5. From the known cDNA of the initial solution and the known sample volume after evaporation, the final cDNA could be estimated to an accuracy of ±5%. The capillaries were sealed when the DNA concentration was in the range cDNA = 260−290 mg/ml, ∼10 mg/ml above the range of the Sm-A phase previously investigated [6]. They were then stored at 4 ◦ C for several weeks and monitored optically in order to ensure uniformity prior to measurements. Polarizing optical microscopy was performed on samples of ∼5−10 μm thickness sandwiched between microscope slides. The samples were sealed by a thin ring of mineral oil after water had evaporated from the edges of the film to the point where focal conic (FC) textures appeared, signaling the development of a smectic phase. To confirm that the orientation of the GDNA duplex segments in the FCs is consistent with a smectic layer structure, we utilized a wedge-shaped quartz compensator plate that could be inserted into the optical path between crossed polarizer and analyzer at a 45◦ angle to the polarizer axis. By sliding the plate a variable distance into the optical path, the phase shift between light polarized parallel and perpendicular to the optic axis of the plate could be varied. The variable retardation, combined with the birefringence and dispersion of the GDNA sample, enables one to determine the orientation of the optic axis in the FC domains by referencing the color of light transmitted through the sample to a Michel-Levy interference color chart [18]. SAXS/WAXS measurements were carried out on beamline 11-BM at the National Synchrotron Light Source II. The incident x-ray energy was 17 keV, the incident beam size at the sample was 0.2 × 0.2 mm, and the SAXS/WAXS area detectors were positioned 3.0 and 0.26 m from the sample, respectively. Samples were mounted in a modified commercial hot/cold stage (Instec HCS60) with kapton film windows. The temperature of the sample was regulated between 5 and 65 ◦ C (safely below the 77 ◦ C denaturation temperature of the 48 bp GDNA duplexes). No evidence of x-ray damage to samples was observed during stepwise temperature scans with 10−30 s exposures at each step. III. RESULTS A. Optical microscopy Smectic layering in GDNA samples is revealed in the polarizing microscope by a FC “fan” texture in which the duplexes orient radially away from the core of individual FCs. As described in Experimental Details, the orientation may be deduced using an optical compensation plate. In order to minimize the cost in elastic energy (i.e., preserve approximately uniform layer spacing), the smectic layers of GDNA bend azimuthally around the FC core, and there is a corresponding splay in the duplex orientation. (By contrast, fully paired duplexes in the columnar phase orient azimuthally within similarly appearing FC domains [3] and thus undergo an orientational bend in these domains). Figure 1 shows two variants of the fan texture coexisting at 25 ◦ C in a thin film of 48-10T-48 sandwiched between glass slides and sealed as described above. The DNA concentration increases from left to right. The dashed yellow line tracks a boundary between smooth (lower cDNA ) and striated (higher cDNA ) fan textures to the left and right of the line, respectively. The images at the bottom of Fig. 1 show details of smooth and striated FCs with different “fan” orientations, before and after the compensator plate was inserted. (Its optical axis is labeled e in the figure.) After the compensator is inserted, the color shifts (relative to background color) observed between “fans” oriented along and perpendicular to e, together with the negative optical anisotropy of DNA duplexes [19–21], confirm a radial duplex orientation within the FCs. We previously used the same technique to associate the smooth fan texture with the Sm-A phase in GDNA [6]. As water evaporates from the sample edge, the smooth texture appears first and then evolves after further evaporation to the 033046-2 SMECTIC-B PHASE AND TEMPERATURE-DRIVEN … PHYSICAL REVIEW RESEARCH 4, 033046 (2022) FIG. 2. Top left and right: 2D SAXS patterns recorded on oriented domains of a 265 mg/ml 48-10T-48 sample in the Sm-B phase at 15.3 ◦ C (left) and in the Sm-A phase at 52.8 ◦ C (right). The central image in each case shows the small angle diffraction arising from layering of the duplex segments (00l peaks, with the l = 1, 2, 4 orders indicated with dashed blue circles for reference). The left and right side images are blow-ups of the wider angle scattering due to the lateral packing of the duplexes within the layers. In the Sm-B phase, this scattering consists of narrowly spaced, sharp arcs indicated by the blue dashed lines, whose index is 10l (l = 0 dominating, with much weaker features for l = 2, 3, 4 being just visible). In the Sm-A phase, the arcs are replaced by diffuse scattering. Bottom left and right: 2D WAXS patterns recorded in the Sm-B and Sm-A phases simultaneously with the SAXS patterns. In the Sm-B phase (bottom left), the dashed blue arcs indicate 100 and (much weaker) 110 peaks indexed for a 2D hexagonal lattice. Bottom middle: Log of azimuthally averaged x-ray intensity vs q obtained from the SAXS pattern at top left (Sm-B phase), with six 00l and three resolvable 10l reflections labeled. The SAXS and WAXS patterns are rendered after the log of the pixel values was taken (with a small offset). The diffuse ring in the WAXS images just beyond the 100 peak is scattering from kapton windows. striated texture, which migrates inward from the edge. It is, therefore, reasonable to attribute the striated texture, developing at higher DNA concentration than the Sm-A, to a distinct, higher-order smectic phase. In small molecule thermotropic LCs, a similar textural change occurs at a temperature-driven Sm-A to a Sm-B phase transition [22], although in that case the stripes decorating the FC fans fade away as the Sm-B order equilibrates thermally. To confirm the nature of the higher-order phase in GDNA and to explore its temperature dependence, we turn to the results of SAXS/WAXS measurements. B. SAXS and WAXS scattering Figure 2 shows representative 2D SAXS/WAXS patterns from fairly well-oriented smectic domains (with layer normal nearly parallel to the detector plane) in a 265 mg/ml 48-10T48 sample at temperatures T = 15.3 and 52.8 ◦ C. At the lower temperature, six orders of diffraction are recorded at small angles (Fig. 2, top left). The lowest order (at scattering wave- number q = 0.187 nm−1 ) corresponds to a spatial periodicity of dB = 33.6 nm, slightly greater than two duplex lengths. We index the peaks as 00l with corresponding scattering vector q00l along the layer normal. Their number and full width at half maximum (FWHM) indicate domains with a well-defined layer structure of average length of at least 30 duplexes along the layer normal. The alternating heights of the odd and even order peaks (Fig. 2, bottom middle) suggest a combination of mono- and bilayer components, with layer spacing dB /2 and dB , respectively, contributing to the overall smectic density wave. At wider angles on the SAXS detector, intense scattering, centered azimuthally about the direction orthogonal to the smectic layer normal, is recorded at q = 2.19 nm−1 for the lower temperature (top left in Fig. 2). The sharpness of the peak and its orientation indicate significant positional correlations of the duplexes within the smectic layers. Weaker reflections, at 2.22, 2.26, and 2.31 nm−1 (narrowly spaced from the main peak and indicated by blue dashes in Fig. 2), are also visible. Additionally, as shown in Fig. 2 (bottom 033046-3 PRABESH GYAWALI et al. PHYSICAL REVIEW RESEARCH 4, 033046 (2022) FIG. 3. Temperature dependence of the log of the azimuthally averaged SAXS intensity from a 285 mg/ml 48-10T-48 GDNA sample. The left panel shows the diffraction from the smectic layer structure with peaks indexed as 00l in the Sm-B phase and 00l  in the Sm-A phase, and the right panel shows a detail of 100 peak diminishing with increasing temperature. Traces in red correspond to heating from the Sm-B to -A phase, and the traces in blue are obtained after subsequent cooling back to the Sm-B. left), a very weak peak, also centered on the axis perpendicular to the layering direction, is recorded on the WAXS detector at q = 3.79 nm−1 . The ratio of wave numbers for the intense and weak WAXS peaks, 3.79/2.19 = 1.73, matches the expected relation between the 110 and the 100 reflec√ tions from a hexagonal crystal structure, q110 /q100 = 3. Based on these assignments, we may index the weaker satellite reflections at 2.22, 2.26, and 2.31 nm−1 √as 10l for l = 2 2 2, 3, 4, since they satisfy the relation q10l = q100 + q00l = √ 2 2 2.19 + (l × 0.187) . As indicated by nearly overlapping blue dashed lines in Fig. 2 (top left), the peak “expected” at q101 = 2.20 nm−1 is too close to the intense scattering at q100 = 2.19 nm−1 to be resolved. Based on the analysis and assignments above, we identify the lower temperature smectic phase as a Sm-B phase, with layers of hexagonally packed 48 bp duplex segments of the GDNA constructs stacked so that their blunt ends are paired, producing a ∼2 duplex periodicity along the stacking √ direction. The lateral duplex-duplex spacing is a = 4π /( 3q100 ) = 3.31 nm (center to center), and the typical lateral size of a monodomain (estimated from the FWHM of the 100 peak) is 25a. The Sm-B state of GDNA is strongly sensitive to temperature as well as concentration. At elevated temperatures, it melts reversibly into a Sm-A phase. In particular, during heating from room temperature, the intense 100 scattering diminishes continuously and, together with the weak 110 peak, disappears completely between 50 and 60 ◦ C, leaving only diffuse scattering from the lateral packing of the duplexes consistent with fluid smectic layers (Fig. 2, top right). At the same temperature, the 00l peaks for odd l (associated with ∼two duplex periodicity) also vanish, resulting in a Sm-A phase with ∼single duplex layer spacing. Detailed SAXS data vs temperature reveal a temperature range over which the Sm-B phase coexists with the Sm-A phase. Figure 3 (left panel) shows azimuthally averaged profiles of x-ray diffraction at small angles and various T from the smectic layer structure of a 48-10T-48 sample with cDNA = 285 mg/ml. At T ≈ 43 ◦ C, a secondary peak emerges at a q value just above the 002 peak associated with the low-temperature (Sm-B) layer structure. This new peak (designated 002 ) and its weaker harmonic (004 ) grow in amplitude as the scattering from Sm-B domains diminishes and eventually (at T ≈ 59 ◦ C) account for all the observed small-angle scattering. Figure 3 (right panel) shows the simultaneous diminution of the wider angle 100 peak on heating through the same temperature steps and its replacement with a broad, diffuse peak. Thus after heating to 59 ◦ C, the Sm-B phase in the 285 mg/ml sample has completely transformed to a Sm-A state, with layer spacing dA = 2π /q002 = 16.6 nm comparable to a single duplex length. When the sample is subsequently cooled back to 22 ◦ C, the Sm-B phase reforms with the 00l (l = 1−6) and 100 peaks reappearing at their original positions (Fig. 3, blue traces), although the amplitudes do not fully recover over a 1-h period after cooling. The Sm-B to -A transition occurs at a ∼7 ◦ C higher temperature in the 285 mg/ml sample than in the more dilute (265 mg/ml) sample studied in Fig. 2. The dependence of the transition temperature on cDNA appears to be significant, but so far we have not explored it in detail. IV. DISCUSSION In addition to hexagonal lateral packing of the duplexes, a model of the Sm-B phase in GDNA must account for both mono- and bilayer correlations, as evidenced by the presence 033046-4 SMECTIC-B PHASE AND TEMPERATURE-DRIVEN … PHYSICAL REVIEW RESEARCH 4, 033046 (2022) FIG. 4. Proposed schemes for GDNA packing in the smectic-A (left) and -B (middle and right) phases of 48-10T-48 GDNA solutions. On the left, right, and back sides of the domains shown, the duplexes are depicted as long, light-blue cylinders, the regions occupied by the ss “gap” segments are shown as short cylinders in a bolder blue, and the blunt duplex ends are highlighted in dark blue. Inside the domains, the constructs are rendered with transparency to reveal the interior arrangement. Partial layers of duplexes are shown at the top and bottom of the domain. Left: In the Sm-A phase, the blunt duplex ends are separated, allowing the ss segments to explore space and the duplexes to disorder within the layer planes. Interdigitation of duplexes from neighboring constructs results in a smectic density wave with ∼single duplex periodicity. Middle: Sm-B phase with hexagonally packed duplexes in the layers. Paired blunt ends and ss segments microphase separate within the planes between the layers, leading to a superposition of density variations along the layer normal with ∼one and ∼two duplex periodicity and a small-angle diffraction profile consistent with the alternating peak intensities in Fig. 3 at low temperature. Right: 3D ordered Sm-B phase with full segregation of paired blunt ends and ss segments into alternating planes, producing pure bilayer stacking. The harmonics in the corresponding diffraction profile should then decrease monotonically with q (which differs from the data in Fig. 3). of odd and even 00l orders of diffraction that alternate systematically in intensity and by the disappearance of the odd orders at the Sm-B to -A transition (Figs. 2 and 3). Figure 4 presents schemes for the GDNA packing in the two phases that satisfy these requirements. The main idea is that the Sm-B phase forms from a lower concentration (or higher temperature) Sm-A phase in which the duplex segments from different GDNA constructs interdigitate within the smectic layers, and their blunt ends are generally separated. As suggested in Fig. 4, this arrangement allows the flexible ss “gap” segments to explore additional space laterally while maintaining a relatively dense packing of duplexes in fluid monolayers (layer spacing comparable to a single duplex length). In this scenario, entropy prevails over enthalpic endend interactions in minimizing the free energy. At higher cDNA (or lower temperature), the Sm-B phase forms as the blunt ends of the duplex segments pair, and the duplexes pack side by side in a 2D hexagonal lattice. The attractive interaction between blunt ends—stronger at lower temperature and higher cDNA —reduces the free energy against a trade-off in entropy. The blunt end pairing eliminates lateral space available for the flexible ss segments to explore, and consequently these extend along the layer normal, expanding the layer spacing. This accounts for the shift (Fig. 3) of the 002 and 004 peaks in the Sm-A phase to their positions at slightly lower q in the -B phase. The development from purely monolayer to substantially bilayer correlations across the SmA to -B transition can be viewed in terms of phase separation of a mixture of blunt duplex ends (shown in dark blue in Fig. 4) and “dressed” ends (i.e., ends connected to a flexible ss, rendered in lighter blue) that occupy planes parallel to the layers. From left to right, Fig. 4 displays a sequence from a disordered, homogeneous mixture within each plane to complete segregation of blunt and “dressed” duplex ends into alternating planes with a hexagonal lateral placement. The middle scheme depicts an intermediate state where blunt and “dressed” end-rich domains coexist in each plane (analogous to two phase coexistence in a binary mixture). The smectic density wave in this case combines bilayer (spacing dB ) and monolayer (spacing dB /2) components, described by Fourier series with harmonics at 2π l/dB for any integer l in the first case and for even l only in the second. This could produce the pattern of 00l diffraction peaks with alternating heights seen in Fig. 3. In the Sm-B phase, pairing of the blunt ends and confinement of the ss connecting the opposing (internal) ends imply a degree of interlayer coupling of the 2D hexagonal ordering, which in turn could explain the weak 10l satellites to the 100 peak (Fig. 2, top left). On the other hand, the weak 110 scattering implies that the 2D hexagonal order within Sm-B layers of GDNA is not as well developed as in the hexagonal 033046-5 PRABESH GYAWALI et al. PHYSICAL REVIEW RESEARCH 4, 033046 (2022) crystalline phase of fully paired duplexes (which occurs at significantly higher cDNA ) [11]. Lastly, we consider the mechanism for the striated optical texture observed on FCs in smectic GDNA samples (Fig. 1). Below the Sm-A to -B transition the lateral spacing (a) between duplexes shrinks (compare centers of the diffuse Sm-A and sharp Sm-B peaks in Fig. 3, right), while the smectic layer spacing increases at the same time. Assuming this also happens when the transition occurs due to increasing cDNA (at constant T ), the lateral strain imposed by reduction in a could be relieved by a bend modulation in the duplex orientation along the layer normal, which is not inhibited by a fixed layer spacing because it is simultaneously changing. The accompanying modulation of the optical birefringence would then produce a stripe pattern, with the stripes parallel to the layers (i.e., running azimuthally across the “fan backs” in the optical texture). If the Sm-B phase equilibrates slowly (due to slow water evaporation and relaxation of concentration gradients), the stripe pattern may persist for long times. the smectic-A phase previously discovered in this system, and a temperature-driven Sm-B to Sm-A transition occurs at fixed concentration when the sample is heated a few tens of degrees above room temperature. These results further support the conclusion that the phase diagram of DNA duplexes in the concentration range ∼200−300 mg/ml, where LC phases occur, shifts from exclusively cholesteric/columnar to predominantly layered (smectic) phases when a sufficiently long segment of unpaired bases is introduced in the middle of an otherwise fully paired construct. The flexible internal linkage and the blunt end-end attractive interaction both play key roles in stabilizing the smectic phases and determining their remarkable combination of thermotropic and lyotropic behavior. 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