1,720,974 research outputs found
EXPLORING THE ROLE OF LIQUID CRYSTAL ORDERING OF DNA OLIGOMERS IN THE PREBIOTIC SYNTHESIS OF NUCLEIC ACIDS
Full text linkThe work in this thesis has been devoted to study the self-assembly of DNA oligomers in light of their possible relevance in the context of the formation of longer chains of nucleic acids in the prebiotic world. This conjecture is based on previous evidence of hierarchical self-assembly of short oligonucleotides in solution that provides mechanisms of self-selection. In such self-assembled structures, the 3’ and 5’ terminals are held in close contact, a condition that could act as a spontaneous template for the elongation of the chains in conditions favoring their chemical ligation.
The work here described was aimed at testing this notion by various investigations targeting what appeared to be the most critical issues in this context. Accordingly, I acted in three main directions.
First I investigated if LC ordering, and hence ligation templating, emerges even in solutions of oligomers having sequences chosen at random, and I determined a new extended phase diagram which includes the degree of randomness. Second, I determined what is the minimum oligomer length which enables the formation of LC phases and I found that LC phases can be found in solution of oligomers as short as 4-bases, and even with randomly chosed sequences. This last result was actually quite suprising and indicates the existence of a new regime for the self-assembly of ultra-short DNA chains. Third, I explored the influence of LC ordering of short DNA oligomers on non-enzymatic ligation reaction favored by the presence of a water-soluble condensing agent. I found a good yield for the polymerization of DNA oligomers both in isotropic than in LC phases, and polymerized chains up to 12 times longer than the initial length.
I believe the work described in this thesis strengthens the notion that self-assembly of nucleic acids could indeed have been the key factor promoting the formation of long chains. These results shad a new light on the most obscure among the processes that enabled the emergence of life on the early Earth
The prebiotic role of Liquid Crystal self-assembly of DNA oligomers
No full textMolecular crowding has a crucial role in tuning the hierarchical self-assembly of complex macromolecular structures and in boosting chemical reactions, allowing the proximity of reactants.
It has been recently shown that at high concentration (> 200 mg/ml) short DNA oligomers (4-20 bases) associated in double helices may order into Liquid Crystal (LC) phases despite their nearly globular shape [1]. In these systems the formation of LC is mediated by the end-to-end aggregation of DNA duplexes into columns of chemically distinct but physically continuous duplexes.
LC ordering of DNA oligomers appears to be a robust phenomenon even in crowded molecular mixtures. Indeed LC phases are found in concentrated solution of random sequence DNA oligomers [2], and in systems in which double stranded DNA is mixed with DNA single strands or with poly-(ethylene glycol) (PEG) chains [3]. In these systems the formation of LC domains is associated with phase separations providing a mechanism of self-selection and compartmentalization of DNA in water, otherwise unusual without the presence of vesicle membrane (see cartoons in Fig.). Moreover we found that DNA-PEG phase separation influences the quality and the yield of non-enzymatic ligation of DNA duplexes, catalyzing the formation of longer strands (graph in Fig.). 1) M. Nakata et al.,Science, 318, 1276 (2007).
2) T. Bellini et al., PNAS, 109, 1110 (2012).
3) G. Zanchetta, M. Nakata, M. Buscaglia, T. Bellini, N.A. Clark, PNAS, 105, 1111 (2008)
Evidence of Liquid Crystal–Assisted Abiotic Ligation of Nucleic Acids
No full textThe emergence of early life must have been marked by the appearance in the prebiotic era of complex molecular structures and systems, motivating the investigation of conditions that could not only facilitate appropriate chemical synthesis, but also provide the mechanisms of molecular selection and structural templating necessary to pilot the complexification toward specific molecular patterns. We recently proposed and demonstrated that these functions could be afforded by the spontaneous ordering of ultrashort nucleic acids oligomers into Liquid Crystal (LC) phases. In such supramolecular assemblies, duplex-forming oligomers are held in average end-to-end contact to form chemically discontinuous but physically continuous double helices. Using blunt ended duplexes, we found that LC formation could both provide molecular selection mechanisms and boost inter-oligomer ligation. This paper provides an essential extension to this notion by investigating the catalytic effects of LC ordering in duplexes with mutually interacting overhangs. Specifically, we studied the influence of LC ordering of 5’-hydroxy-3’-phosphate partially self-complementary DNA 14mers with 3’-CG sticky-ends, on the efficiency of non-enzymatic ligation reaction induced by water-soluble carbodiimide EDC as condensing agent. We investigated the ligation products in mixtures of DNA with poly-ethylene glycol (PEG) at three PEG concentrations at which the system phase separates creating DNA-rich droplets that organize into isotropic, nematic LC and columnar LC phases. We observe remarkable LC-enhanced chain lengthening, and we demonstrate that such lengthening effectively promotes and stabilizes LC domains, providing the kernel of a positive feedback cycle by which LC ordering promotes elongation, in turn stabilizing the LC ordering
Liquid Crystal Coacervates Composed of Short Double-Stranded DNA and Cationic Peptides
Full text linkPhase separation of nucleic acids and proteins is a ubiquitous phenomenon regulating subcellular compartment structure and function. While complex coacervation of flexible single-stranded nucleic acids is broadly investigated, coacervation of double-stranded DNA (dsDNA) is less studied because of its propensity to generate solid precipitates. Here, we reverse this perspective by showing that short dsDNA and poly-L-lysine coacervates can escape precipitation while displaying a surprisingly complex phase diagram, including the full set of liquid crystal (LC) mesophases observed to date in bulk dsDNA. Short dsDNA supramolecular aggregation and packing in the dense coacervate phase are the main parameters regulating the global LC-coacervate phase behavior. LC-coacervate structure was characterized upon variations in temperature and monovalent salt, DNA, and peptide concentrations, which allow continuous reversible transitions between all accessible phases. A deeper understanding of LC-coacervates can gain insights to decipher structures and phase transition mechanisms within biomolecular condensates, to design stimuli-responsive multiphase synthetic compartments with different degrees of order and to exploit self-assembly driven cooperative prebiotic evolution of nucleic acids and peptides
Liquid Crystal Peptide/DNA Coacervates in the Context of Prebiotic Molecular Evolution
Full text linkLiquid–liquid phase separation (LLPS) phenomena are ubiquitous in biological systems, as various cellular LLPS structures control important biological processes. Due to their ease of in vitro assembly into membraneless compartments and their presence within modern cells, LLPS systems have been postulated to be one potential form that the first cells on Earth took on. Recently, liquid crystal (LC)-coacervate droplets assembled from aqueous solutions of short double-stranded DNA (s-dsDNA) and poly-L-lysine (PLL) have been reported. Such LC-coacervates conjugate the advantages of an associative LLPS with the relevant long-range ordering and fluidity properties typical of LC, which reflect and propagate the physico-chemical properties of their molecular constituents. Here, we investigate the structure, assembly, and function of DNA LC-coacervates in the context of prebiotic molecular evolution and the emergence of functional protocells on early Earth. We observe through polarization microscopy that LC-coacervate systems can be dynamically assembled and disassembled based on prebiotically available environmental factors including temperature, salinity, and dehydration/rehydration cycles. Based on these observations, we discuss how LC-coacervates can in principle provide selective pressures effecting and sustaining chemical evolution within partially ordered compartments. Finally, we speculate about the potential for LC-coacervates to perform various biologically relevant properties, such as segregation and concentration of biomolecules, catalysis, and scaffolding, potentially providing additional structural complexity, such as linearization of nucleic acids and peptides within the LC ordered matrix, that could have promoted more efficient polymerization. While there are still a number of remaining open questions regarding coacervates, as protocell models, including how modern biologies acquired such membraneless organelles, further elucidation of the structure and function of different LLPS systems in the context of origins of life and prebiotic chemistry could provide new insights for understanding new pathways of molecular evolution possibly leading to the emergence of the first cells on Earth
Random-Sequence DNA Oligomers Make Liquid Crystals: A Case of Collective Ordering in a Superdiverse Environment
No full textThe availability of synthetic, analytical, and predictive tools makes DNA an ideal platform, not yet considered, to investigate statistical and soft matter physics. Here we report and interpret the equilibrium collective ordering in solutions of random-sequence DNA (rsDNA) oligomers of lengths L = 8 and L = 12. Despite the extreme molecular diversity inherent in rsDNA solutions, which for L = 12 are composed of 20 million distinct molecular species, these systems develop long-range columnar liquid crystal (LC) ordering when equilibrated at high osmotic pressure. By a combination of experimental models and computed statistics, we demonstrate that the residual end-to-end attraction between rsDNA duplexes, which typically terminate with various forms of pairing errors, is indeed sufficient to drive LC ordering. The resulting narrow range of isotropic-LC phase coexistence, in seeming contrast with the variety of phase behaviors of the species composing rsDNA, demonstrates that the (nearly) continuum distribution of molecular interaction strengths effectively reduces the tendency for demixing instead of enhancing it, in line with theoretical modeling
Liquid Crystal ordering of DNA Dickerson Dodecamer duplexes with different 5’- Phosphate terminations
Full text linkThe onset of liquid crystal (LC) phases in concentrated aqueous solutions of DNA oligomers crucially depends on the end-to-end interaction between the DNA duplexes, which can be provided by the aromatic stacking of the terminal base-pairs or by the pairing of complementary dangling-ends. Here we investigated the LC behavior of three blunt-end 12-base-long DNA duplexes synthesized with hydroxyl, phosphate and triphosphate 5'-termini. We experimentally characterized the concentration-temperature phase diagrams and we quantitatively estimated the end-to-end stacking free energy, by comparing the empirical data with the predictions of coarse-grained linear aggregation models. The preservation of LC ordering, even in presence of the bulky and highly charged triphosphate group, indicates that attractive stacking interactions are still present and capable of induce linear aggregation of the DNA duplexes. This finding strengthens the potential role of chromonic like self-assembly for the prebiotic formation of linear polymeric nucleic acids
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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