Short complementary B-form DNA oligomers, 6 to 20 base pairs in length, are found to exhibit nematic and columnar liquid crystal phases, even though such duplexes lack the shape anisotropy required for liquid crystal ordering. Structural study shows that these phases are produced by the end-to-end adhesion and consequent stacking of the duplex oligomers into polydisperse anisotropic rod-shaped aggregates, which can order into liquid crystals. Upon cooling mixed solutions of short DNA oligomers, in which only a small fraction of the DNA present is complementary, the duplex-forming oligomers phase-separate into liquid crystal droplets, leaving the unpaired single strands in isotropic solution. In a chemical environment where oligomer ligation is possible, such ordering and condensation would provide an autocatalytic link whereby complementarity promotes the extended polymerization of complementary oligomers.

Concentrated solutions of duplex-forming DNA oligomers organize into various mesophases among which is the nematic (N Ã ), which exhibits a macroscopic chiral helical precession of molecular orientation because of the chirality of the DNA molecule. Using a quantitative analysis of the transmission spectra in polarized optical microscopy, we have determined the handedness and pitch of this chiral nematic helix for a large number of sequences ranging from 8 to 20 bases. The B-DNA molecule exhibits a right-handed molecular double-helix structure that, for long molecules, always yields N Ã phases with left-handed pitch in the μm range. We report here that ultrashort oligomeric duplexes show an extremely diverse behavior, with both left-and right-handed N Ã helices and pitches ranging from macroscopic down to 0.3 μm. The behavior depends on the length and the sequence of the oligomers, and on the nature of the end-to-end interactions between helices. In particular, the N Ã handedness strongly correlates with the oligomer length and concentration. Right-handed phases are found only for oligomers shorter than 14 base pairs, and for the sequences having the transition to the N Ã phase at concentration larger than 620 mg∕mL. Our findings indicate that in short DNA, the intermolecular doublehelical interactions switch the preferred liquid crystal handedness when the columns of stacked duplexes are forced at high concentrations to separations comparable to the DNA double-helix pitch, a regime still to be theoretically described.cholesteric | RNA U nderstanding how the physicochemical details of macromolecules affect their interactions and their self-ordering properties represents one of the major challenges in the physics of soft and biological matter. In particular, because most of the biologically relevant molecules are chiral-i.e., they lack mirror symmetry-the relevant biointeractions are typically highly sensitive to the molecular handedness. One important effect of chiral interactions is the propagation of chirality from the molecular structure to the supramolecular assemblies such as aggregates or ordered phases. Often, in this phenomenon, minor molecular modifications are amplified to produce remarkable changes in the macroscopic ordering, as for DNA supercoiling and its biological role (1). The propagation of chirality has been studied in various systems, including thermotropic liquid crystals (LCs) and lyotropic assemblies of viruses, DNA, polymers, and dye aggregates. Despite the generality of the phenomenon, its understanding is still poor. Predicting, only on the basis of the molecular structure, the most basic parity of chiral LC ordering, the rightvs. left-handedness, is still a challenge (2-4). One would expect the problem to become easier when restricted to molecular structures with a clean cylindrical shape decorated with regular helical structures and charges, such as DNA, G-quartets, and filamentous viruses. However, even in this limited frame, experimental and theoretical results appear difficult to fi...

Abstract:Innovative technical solutions to realize optical biosensors with improved performance are continuously proposed. Progress in material fabrication enables developing novel substrates with enhanced optical responses. At the same time, the increased spectrum of available biomolecular tools, ranging from highly specific receptors to engineered bioconjugated polymers, facilitates the preparation of sensing surfaces with controlled functionality. What remains often unclear is to which extent this continuous innovation provides effective breakthroughs for specific applications. In this review, we address this challenging question for the class of label-free optical biosensors, which can provide a direct signal upon molecular binding without using secondary probes. Label-free biosensors have become a consolidated approach for the characterization and screening of molecular interactions in research laboratories. However, in the last decade, several examples of other applications with high potential impact have been proposed. We review the recent advances in label-free optical biosensing technology by focusing on the potential competitive advantage provided in selected emerging applications, grouped on the basis of the target type. In particular, direct and real-time detection allows the development of simpler, compact, and rapid analytical methods for different kinds of targets, from proteins to DNA and viruses. The lack of secondary interactions facilitates the binding of small-molecule targets and minimizes the perturbation in single-molecule detection. Moreover, the intrinsic versatility of label-free sensing makes it an ideal platform to be integrated with biomolecular machinery with innovative functionality, as in case of the molecular tools provided by DNA nanotechnology.

Two new heteroleptic pentacoordinated Zn(II) complexes (1 and 2) containing 4,4 0 -disubstituted 2,2 0bipyridines as the main ligand and curcumin (curc) as an ancillary ligand have been synthesized, spectroscopically and structurally characterized, and tested in vitro towards different human cancer cell lines. While the nitrogen ligands are almost inactive, Zn(II) curc derivatives 1 and 2 show promising and selective anticancer properties. In particular the curc Zn(II) complex 1 shows the strongest growth inhibition in all cell lines, being even more effective than the pure curc in the LAN-5 neuroblastoma cell line. Furthermore, the curc moiety makes the complexes 1 and 2 fluorescent, a feature enabling investigation of their interaction with DNA through a new optical method previously tested with the reference fluorescent intercalator ethidium bromide. This analysis demonstrates that the interaction mode of curc, 1 and 2 with DNA in the double helix favors their alignment perpendicular to the DNA axis, suggesting a partial inter-base intercalation of these Zn(II) complexes.

Using optical microscopy, we have studied the phase behavior of mixtures of 12-to 22-bp-long nanoDNA oligomers. The mixtures are chosen such that only a fraction of the sample is composed of mutually complementary sequences, and hence the solutions are effectively mixtures of single-stranded and double-stranded (duplex) oligomers. When the concentrations are large enough, such mixtures phase-separate via the nucleation of duplex-rich liquid crystalline domains from an isotropic background rich in single strands. We find that the phase separation is approximately complete, thus corresponding to a spontaneous purification of duplexes from the single-strand oligos. We interpret this behavior as the combined result of the energy gain from the end-to-end stacking of duplexes and of depletion-type attractive interactions favoring the segregation of the more rigid duplexes from the flexible single strands. This form of spontaneous partitioning of complementary nDNA offers a route to purification of short duplex oligomers and, if in the presence of ligation, could provide a mode of positive feedback for the preferential synthesis of longer complementary oligomers, a mechanism of possible relevance in prebiotic environments.condensation ͉ nucleation ͉ depletion ͉ prebiotic ͉ PEG M olecular crowding of cellular interior is known to play a role in the spontaneous organization of biological macromolecules (1). Several biochemical and physiological processes are found to be influenced by strong packing constraints (2, 3), and complex ordered arrangements, such as liquid-crystalline mesophases, have been shown to arise from highly packed biomolecules (4). Of particular interest is the ordering of highly concentrated DNA in cell nuclei, which can lead to a variety of mesophases in vivo (5). However, whether such ordering caused by packing constraints had represented an evolutionary advantage remains an open question.Concentrated solutions of fully hybridized nanoDNA (nDNA) exhibit various liquid crystalline forms of supramolecular ordering, promoted by end-to-end adhesion of the paired bases at the terminals of the duplexes. This behavior has been reported recently for a wide set of self-complementary (SC) 6-to 20-bp nDNA sequences and for mutually complementary sequences in the same length range (6). Here we explore the phase behavior of mixtures of nDNA in which only some of the sequences are complementary, thus able to pair in double strands (DSs), and part of the sequences are not, and thus always remain in the solution as single strands (SSs). We have found that in concentrated mixtures of duplex and SS nDNA, the system phaseseparates into duplex-rich liquid crystal (LC) domains coexisting with a duplex-poor isotropic phase, leading to the physical segregation of 4-to 6-nm-long complementary chains from noncomplementary ones. This phase separation is a collective effect of the duplex and SS nDNA because the duplexes alone would not form LCs in such solutions, their concentration being well below that required for LC forma...

In biological systems and nanoscale assemblies, the self-association of DNA is typically studied and applied in the context of the evolved or directed design of base sequences that give complementary pairing, duplex formation, and specific structural motifs. Here we consider the collective behavior of DNA solutions in the distinctly different regime where DNA base sequences are chosen at random or with varying degrees of randomness. We show that in solutions of completely random sequences, corresponding to a remarkably large number of different molecules, e.g., approximately 10 12 for random 20-mers, complementary still emerges and, for a narrow range of oligomer lengths, produces a subtle hierarchical sequence of structured self-assembly and organization into liquid crystal (LC) phases. This ordering follows from the kinetic arrest of oligomer association into long-lived partially paired double helices, followed by reversible association of these pairs into linear aggregates that in turn condense into LC domains.T he selectivity and reversibility of DNA and RNA association enables crucial biological functions in which oligomers selectively pair to target sequences even within large amounts of nucleic acid chains. Selectivity is decisive, for example, in the microRNA-mRNA interactions, crucial in the regulation of gene expression. Similar high levels of selectivity are exploited in genomic PCR, relying on the capacity of primers to target their complementary sequence within a full genome. Selective interactions of DNA oligomers have been exploited in the past years in a variety of strategies for the construction of designed self-assembled nanostructures (1-4). Selectivity combines with self-assembly in the recent observation that short oligomers of nucleic acids having complementary sequences exhibit liquid crystal (LC) ordering (5-7). In this article, we report LC ordering in solutions of DNA oligomers with random sequences where the large body of different competing sequences effectively reduces the selectivity of the interactions. With these results, we show that the phenomenology of the self-assembly of nucleic acid oligomers is actually much richer than previously recognized, involving self-selection, linear aggregation, and ordering of fully random chains. Our results strengthen the notion that DNA and RNA have unequaled capacity of self-structuring and unavoidably suggests self-assembly as the possible key factor for the emergence of nucleic acids from the prebiotic molecular clutter as the coding molecules of life. LC Ordering of Complementary DNA SequencesThe first observations of LC ordering of oligonucleotides were performed in solutions of 6-to 20-base-pair DNA oligomers (6 bp ≤ N B ≤ 20 bp) whose sequences promoted the formation of fully paired duplexes (example 1 in Fig. 1A). These were found to order into the chiral nematic (N Ã ) LC phase in concentration (c DNA ) ranges depending on the oligomer length and sequence. At larger c DNA , the solutions transform into the columnar (COL) phase and, at ...

SARS-CoV-2 symptoms are non-specific and can range from asymptomatic presentation to severe pneumonia. Asymptomatic subjects carrying SARS-CoV-2 often remain undiagnosed and it is still debated whether they develop immunoglobulins (Ig) and how long they persist. The aim of this study was to investigate the development and persistence of antibodies against SARS-CoV-2 in asymptomatic subjects infected by the virus. This follow-up study was performed on the 31 asymptomatic subjects who presented a positive nasal swab or serology against SARS-CoV-2 (Ig against Spike-RBD) in the first part of the UNICORN study (March 2020) aimed at attesting previous or current contacts with the virus in the personnel of the University of Milan. Eight weeks after the first Ig measure, these subjects were invited to donate a second blood sample for testing serum antibodies (IgM, IgG and total antibodies) and to fill-in a structured questionnaire. About 80% of asymptomatic subjects did not present circulating immunoglobulins against SARS-CoV-2 after 8 weeks from a positive nasal swab against the virus. Moreover, in more than 40% of these subjects, no Ig against SARS-CoV-2 were detected at any time. Finally, about two third of subjects with immunoglobulins at baseline did not present IgG against SARS-CoV-2 after 8 weeks. The majority of subjects who developed an asymptomatic SARS-CoV-2 infection do not present antibodies against the RBD-spike protein after 8 weeks of follow-up. These data should be taken into account for the interpretation of the serological evidences on SARS-CoV-2 that are emerging nowadays.

It has been observed that concentrated solutions of short DNA oligomers develop liquid crystal ordering as the result of a hierarchically structured supramolecular self-assembly. In mixtures of oligomers with various degree of complementarity, liquid crystal microdomains are formed via the selective aggregation of those oligomers that have a sufficient degree of duplexing and propensity for physical polymerization. Here we show that such domains act as fluid and permeable microreactors in which the order-stabilized molecular contacts between duplex terminals serve as physical templates for their chemical ligation. In the presence of abiotic condensing agents, liquid crystal ordering markedly enhances ligation efficacy, thereby enhancing its own phase stability. The coupling between order-templated ligation and selectivity provided by supramolecular ordering enables an autocatalytic cycle favouring the growth of DNA chains, up to biologically relevant lengths, from few-base long oligomers. This finding suggests a novel scenario for the abiotic origin of nucleic acids.