Cryo-EM structure and functional landscape of an RNA polymerase ribozyme.

The emergence of an RNA replicase capable of self-replication is considered an important stage in the origin of life. RNA polymerase ribozymes (PR) - including a variant that uses trinucleotide triphosphates (triplets) as substrates - have been created by in vitro evolution and are the closest functional analogues of the replicase, but the structural basis for their function is poorly understood. Here we use single-particle cryogenic electron microscopy (cryo-EM) and high-throughput mutation analysis to obtain the structure of a triplet polymerase ribozyme (TPR) apoenzyme and map its functional landscape. The cryo-EM structure at 5-Å resolution reveals the TPR as an RNA heterodimer comprising a catalytic subunit and a noncatalytic, auxiliary subunit, resembling the shape of a left hand with thumb and fingers at a 70° angle. The two subunits are connected by two distinct kissing-loop (KL) interactions that are essential for polymerase function. Our combined structural and functional data suggest a model for templated RNA synthesis by the TPR holoenzyme, whereby heterodimer formation and KL interactions preorganize the TPR for optimal primer-template duplex binding, triplet substrate discrimination, and templated RNA synthesis. These results provide a better understanding of TPR structure and function and should aid the engineering of more efficient PRs.

Solvent viscosity facilitates replication and ribozyme catalysis from an RNA duplex in a model prebiotic process.

The RNA World hypothesis posits that RNA was once responsible for genetic information storage and catalysis. However, a prebiotic mechanism has yet to be reported for the replication of duplex RNA that could have operated before the emergence of polymerase ribozymes. Previously, we showed that a viscous solvent enables information transfer from one strand of long RNA duplex templates, overcoming 'the strand inhibition problem'. Here, we demonstrate that the same approach allows simultaneous information transfer from both strands of long duplex templates. An additional challenge for the RNA World is that structured RNAs (like those with catalytic activity) function poorly as templates in model prebiotic RNA synthesis reactions, raising the question of how a single sequence could serve as both a catalyst and as a replication template. Here, we show that a viscous solvent also facilitates the transition of a newly synthesized hammerhead ribozyme sequence from its inactive, duplex state to its active, folded state. These results demonstrate how fluctuating environmental conditions can allow a ribozyme sequence to alternate between acting as a template for replication and functioning as a catalyst, and illustrate the potential for temporally changing environments to enable molecular processes necessary for the origin of life.

A viscous solvent enables information transfer from gene-length nucleic acids in a model prebiotic replication cycle.

Many hypotheses concerning the nature of early life assume that genetic information was once transferred through the template-directed synthesis of RNA, before the emergence of coded enzymes. However, attempts to demonstrate enzyme-free, template-directed synthesis of nucleic acids have been limited by 'strand inhibition', whereby transferring information from a template strand in the presence of its complementary strand is inhibited by the stability of the template duplex. Here, we use solvent viscosity to circumvent strand inhibition, demonstrating information transfer from a gene-length template (>300 nt) within a longer (545 bp or 3 kb) duplex. These results suggest that viscous environments on the prebiotic Earth, generated periodically by water evaporation, could have facilitated nucleic acid replication-particularly of long, structured sequences such as ribozymes. Our approach works with DNA and RNA, suggesting that viscosity-mediated replication is possible for a range of genetic polymers, perhaps even for informational polymers that may have preceded RNA.

DNA-Origami-Driven Lithography for Patterning on Gold Surfaces with Sub-10 nm Resolution.

Sub-10 nm lithography of DNA patterns is achieved using the DNA-origami stamping method. This new strategy utilizes DNA origami to bind a preprogrammed DNA ink pattern composed of thiol-modified oligonucleotides on gold surfaces. Upon denaturation of the DNA origami, the DNA ink pattern is exposed. The pattern can then be developed by hybridization with complementary strands carrying gold nanoparticles.

Folding and imaging of DNA nanostructures in anhydrous and hydrated deep-eutectic solvents.

There is great interest in DNA nanotechnology, but its use has been limited to aqueous or substantially hydrated media. The first assembly of a DNA nanostructure in a water-free solvent, namely a low-volatility biocompatible deep-eutectic solvent composed of a 4:1 mixture of glycerol and choline chloride (glycholine), is now described. Glycholine allows for the folding of a two-dimensional DNA origami at 20 °C in six days, whereas in hydrated glycholine, folding is accelerated (≤3 h). Moreover, a three-dimensional DNA origami and a DNA tail system can be folded in hydrated glycholine under isothermal conditions. Glycholine apparently reduces the kinetic traps encountered during folding in aqueous solvent. Furthermore, folded structures can be transferred between aqueous solvent and glycholine. It is anticipated that glycholine and similar solvents will allow for the creation of functional DNA structures of greater complexity by providing a milieu with tunable properties that can be optimized for a range of applications and nanostructures.

Spontaneous prebiotic formation of a ß-ribofuranoside that self-assembles with a complementary heterocycle.

The RNA World hypothesis is central to many current theories regarding the origin and early evolution of life. However, the formation of RNA by plausible prebiotic reactions remains problematic. Formidable challenges include glycosidic bond formation between ribose and the canonical nucleobases, as well as the inability of nucleosides to mutually select their pairing partners from a complex mixture of other molecules prior to polymerization. Here we report a one-pot model prebiotic reaction between a pyrimidine nucleobase (2,4,6-triaminopyrimidine, TAP) and ribose, which produces TAP-ribose conjugates in high yield (60-90%). When cyanuric acid (CA), a plausible ancestral nucleobase, is mixed with a crude TAP+ribose reaction mixture, micrometer-length supramolecular, noncovalent assemblies are formed. A major product of the TAP+ribose reaction is a ß-ribofuranoside of TAP, which we term TARC. This nucleoside is also shown to efficiently form supramolecular assemblies in water by pairing and stacking with CA. These results provide a proof-of-concept system demonstrating that several challenges associated with the prebiotic emergence of RNA, or pre-RNA polymers, may not be as problematic as widely believed.

DNA origami as a DNA repair nanosensor at the single-molecule level.

The folding of DNA molecules by DNA origami is used in a nanosensor to analyze enzymatic DNA repair activity of hAGT. The method uses conformational changes that condition α-thrombin interaction with DNA aptamers, and illustrates the use of DNA origami as a proteinrecognition biosensor.

Efficient self-assembly in water of long noncovalent polymers by nucleobase analogues.

Molecular self-assembly is widely appreciated to result from a delicate balance between several noncovalent interactions and solvation effects. However, current design approaches for achieving self-assembly in water with small, synthetic molecules do not consider all aspects of the hydrophobic effect, in particular the requirement of surface areas greater than 1 nm(2) for an appreciable free energy of hydration. With the concept of a minimum hydrophobic surface area in mind, we designed a system that achieves highly cooperative self-assembly in water. Two weakly interacting low-molecular-weight monomers (cyanuric acid and a modified triaminopyrimidine) are shown to form extremely long supramolecular polymer assemblies that retain water solubility. The complete absence of intermediate assemblies means that the observed equilibrium is between free monomers and supramolecular assemblies. These observations are in excellent agreement with literature values for the free energy of nucleic acid base interactions as well as the calculated free energy penalty for the exposure of hydrophobic structures in water. The results of our study have implications for the design of new self-assembling structures and hydrogel-forming molecules and may provide insights into the origin of the first RNA-like polymers.

Nanotribology results show that DNA forms a mechanically resistant 2D network in metaphase chromatin plates.

In a previous study, we found that metaphase chromosomes are formed by thin plates, and here we have applied atomic force microscopy (AFM) and friction force measurements at the nanoscale (nanotribology) to analyze the properties of these planar structures in aqueous media at room temperature. Our results show that high concentrations of NaCl and EDTA and extensive digestion with protease and nuclease enzymes cause plate denaturation. Nanotribology studies show that native plates under structuring conditions (5 mM Mg2+) have a relatively high friction coefficient (µ≈0.3), which is markedly reduced when high concentrations of NaCl or EDTA are added (µ≈0.1). This lubricant effect can be interpreted considering the electrostatic repulsion between DNA phosphate groups and the AFM tip. Protease digestion increases the friction coefficient (µ≈0.5), but the highest friction is observed when DNA is cleaved by micrococcal nuclease (µ≈0.9), indicating that DNA is the main structural element of plates. Whereas nuclease-digested plates are irreversibly damaged after the friction measurement, native plates can absorb kinetic energy from the AFM tip without suffering any damage. These results suggest that plates are formed by a flexible and mechanically resistant two-dimensional network which allows the safe storage of DNA during mitosis.

Aggregated electronegative low density lipoprotein in human plasma shows a high tendency toward phospholipolysis and particle fusion.

Aggregation and fusion of lipoproteins trigger subendothelial retention of cholesterol, promoting atherosclerosis. The tendency of a lipoprotein to form fused particles is considered to be related to its atherogenic potential. We aimed to isolate and characterize aggregated and nonaggregated subfractions of LDL from human plasma, paying special attention to particle fusion mechanisms. Aggregated LDL was almost exclusively found in electronegative LDL (LDL(-)), a minor modified LDL subfraction, but not in native LDL (LDL(+)). The main difference between aggregated (agLDL(-)) and nonaggregated LDL(-) (nagLDL(-)) was a 6-fold increased phospholipase C-like activity in agLDL(-). agLDL(-) promoted the aggregation of LDL(+) and nagLDL(-). Lipoprotein fusion induced by α-chymotrypsin proteolysis was monitored by NMR and visualized by transmission electron microscopy. Particle fusion kinetics was much faster in agLDL(-) than in nagLDL(-) or LDL(+). NMR and chromatographic analysis revealed a rapid and massive phospholipid degradation in agLDL(-) but not in nagLDL(-) or LDL(+). Choline-containing phospholipids were extensively degraded, and ceramide, diacylglycerol, monoacylglycerol, and phosphorylcholine were the main products generated, suggesting the involvement of phospholipase C-like activity. The properties of agLDL(-) suggest that this subfraction plays a major role in atherogenesis by triggering lipoprotein fusion and cholesterol accumulation in the arterial wall.

Dense chromatin plates in metaphase chromosomes.

In a previous work we observed multilayered plate-like structures surrounding partially denatured HeLa chromosomes at metaphase ionic conditions. This unexpected finding has led us to carry out an extensive investigation of these structures. Our results show that plates can also be found in metaphase chromosomes from chicken lymphocytes. We have used atomic force microscopy (AFM) to image and investigate the mechanical properties of plates in aqueous solution. Plates are thin (approximately 6.5 nm each layer) but compact and resistant to penetration by the AFM tip: their Young's modulus is approximately 0.2 GPa and the stress required for surface penetration is approximately 0.03 GPa in the presence of Mg(2+) (5-20 mM). Low-ionic strength conditions produce emanation of chromatin fibers from the edges of uncrosslinked plates. These observations and AFM results obtained applying high forces indicate that the chromatin filament is tightly tethered inside the plates. Images of metal-shadowed plates and cryo-electron microscopy images of frozen-hydrated plates suggest that nucleosomes are tilted with respect to the plate surface to allow an interdigitation between the successive layers and a thickness reduction compatible with the observed plate height. The similarities between denatured plates from chicken chromosomes and aggregates of purified chromatin from chicken erythrocytes suggest that chromatin has intrinsic structural properties leading to plate formation. Scanning electron micrographs and images obtained with the 200-kV transmission microscope show that plates are the dominant component of compact chromatids. We propose that metaphase chromosomes are formed by many stacked plates perpendicular to the chromatid axis.

Structural elements of bulk chromatin within metaphase chromosomes.

We have performed a very extensive electron microscopy investigation of the chromatin structures extruded from partially denatured metaphase chromosomes from HeLa cells under a wide variety of conditions. Denatured chromosomes having fibres as the dominant structural element are obtained in the presence of buffers of very low concentration or after incubation with water. At slightly higher ionic concentrations, metaphase chromosomes become granulated. The most frequently observed granules have a diameter of about 35 nm and show the same structural characteristics as the compact cylindrical chromatin bodies previously found in our laboratory in studies performed using small chromatin fragments. Our results suggest that fibres are formed by the face-to-face association of 35-nm chromatin bodies. We have observed a very compact morphology of chromosomes in solutions containing intracellular concentrations of monovalent cations and the Mg2+ concentration found in metaphase. The most abundant structural elements observed in chromatin extruded from partially denatured compact metaphase chromosomes are multilayered plate-like structures. This is the first time that these planar structures have been reported. The observation of the irregular plates found in some preparations and of the small planar structures seen in aggregates of small chromatin fragments suggests that plates are formed by side-by-side association of compact chromatin bodies.