Free-Energy Landscape and Characteristic Forces for the Initiation of DNA Unzipping

DNA unzipping, the separation of its double helix into single strands, is crucial in modulating a host of genetic processes. Although the large-scale separation of double-stranded DNA has been studied with a variety of theoretical and experimental techniques, the minute details of the very first steps of unzipping are still unclear. Here, we use atomistic molecular-dynamics simulations, coarse-grained simulations, and a statistical-mechanical model to study the initiation of DNA unzipping by an external force. Calculation of the potential of mean force profiles for the initial separation of the first few terminal basepairs in a DNA oligomer revealed that forces ranging between 130 and 230 pN are needed to disrupt the first basepair, and these values are an order of magnitude larger than those needed to disrupt basepairs in partially unzipped DNA. The force peak has an echo of ∼50 pN at the distance that unzips the second basepair. We show that the high peak needed to initiate unzipping derives from a free-energy basin that is distinct from the basins of subsequent basepairs because of entropic contributions, and we highlight the microscopic origin of the peak. To our knowledge, our results suggest a new window of exploration for single-molecule experiments.     

Dynamics of the DNA duplex formation studied by single molecule force measurements

DNA is partly denatured in vitro by applying a force that mechanically separates the two strands of the double helix. Sudden reduction of the imposed displacement triggers spontaneous reannealing of the molecule. The corresponding force signals are measured by optical trapping interferometry for backward steps of various amplitudes and base sequence intervals. The measured signals frequently show plateaus of varying duration at discrete values that are dependent on the base sequence. Additional measurements are performed with proteins bound to the double helix. When the opening fork encounters such a protein during mechanical unzipping, force increases until the protein is ejected. This ejection induces fast release of tension and fast unzipping. Comparing our different measurements, we find that both DNA unzipping and the relaxation of tension in DNA are faster than the formation of the double helix.     

The Origins of Cellular Life

Understanding the origin of cellular life on Earth requires the discovery of plausible pathways for the transition from complex prebiotic chemistry to simple biology, defined as the emergence of chemical assemblies capable of Darwinian evolution. We have proposed that a simple primitive cell, or protocell, would consist of two key components: a protocell membrane that defines a spatially localized compartment, and an informational polymer that allows for the replication and inheritance of functional information. Recent studies of vesicles composed of fatty-acid membranes have shed considerable light on pathways for protocell growth and division, as well as means by which protocells could take up nutrients from their environment. Additional work with genetic polymers has provided insight into the potential for chemical genome replication and compatibility with membrane encapsulation. The integration of a dynamic fatty-acid compartment with robust, generalized genetic polymer replication would yield a laboratory model of a protocell with the potential for classical Darwinian biological evolution, and may help to evaluate potential pathways for the emergence of life on the early Earth. Here we discuss efforts to devise such an integrated protocell model.The emergence of the first cells on the early Earth was the culmination of a long history of prior chemical and geophysical processes. Although recognizing the many gaps in our knowledge of prebiotic chemistry and the early planetary setting in which life emerged, we will assume for the purpose of this review that the requisite chemical building blocks were available, in appropriate environmental settings. This assumption allows us to focus on the various spontaneous and catalyzed assembly processes that could have led to the formation of primitive membranes and early genetic polymers, their coassembly into membrane-encapsulated nucleic acids, and the chemical and physical processes that allowed for their replication. We will discuss recent progress toward the construction of laboratory models of a protocell (Fig. 1), evaluate the remaining steps that must be achieved before a complete protocell model can be constructed, and consider the prospects for the observation of spontaneous Darwinian evolution in laboratory protocells. Although such laboratory studies may not reflect the specific pathways that led to the origin of life on Earth, they are proving to be invaluable in uncovering surprising and unanticipated physical processes that help us to reconstruct plausible pathways and scenarios for the origin of life.Open in a separate windowFigure 1.A simple protocell model based on a replicating vesicle for compartmentalization, and a replicating genome to encode heritable information. A complex environment provides lipids, nucleotides capable of equilibrating across the membrane bilayer, and sources of energy (left), which leads to subsequent replication of the genetic material and growth of the protocell (middle), and finally protocellular division through physical and chemical processes (right). (Reproduced from Mansy et al. 2008 and reprinted with permission from Nature Publishing ©2008.)The term protocell has been used loosely to refer to primitive cells or to the first cells. Here we will use the term protocell to refer specifically to cell-like structures that are spatially delimited by a growing membrane boundary, and that contain replicating genetic information. A protocell differs from a true cell in that the evolution of genomically encoded advantageous functions has not yet occurred. With a genetic material such as RNA (or perhaps one of many other heteropolymers that could provide both heredity and function) and an appropriate environment, the continued replication of a population of protocells will lead inevitably to the spontaneous emergence of new coded functions by the classical mechanism of evolution through variation and natural selection. Once such genomically encoded and therefore heritable functions have evolved, we would consider the system to be a complete, living biological cell, albeit one much simpler than any modern cell (Szostak et al. 2001).     

The evolutionary sequence: origin and emergences

The evolutionary sequence is being reexamined experimentally from a "Big Bang"origin to the protocell and from the emergence of protocell and variety of species to Darwin's mental power (mind) and society (The Descent of Man). A most fundamentally revisionary consequence of experiments is an emphasis on endogenous ordering. This principle, seen vividly in ordered copolymerization of amino acids, has had new impact on the theory of Darwinian evolution and has been found to apply to the entire sequence. Herein, I will discuss some problems of dealing with teaching controversial subjects.     

Stability decrease of RNA double helices by phenylalanine-, tyrosine- and tryptophane-amides. Analysis in terms of site binding and relation to melting proteins.

The amides of L-phenylalanine, L-tyrosine and L-tryptophane decrease the melting temperatures tm of poly(A)*poly(U) and poly(I)*poly(C) double helices at low concentrations (1 mM), whereas high concentrations finally lead to an increase of tm. This dependence of the tm-values upon the ligand concentration can be represented quantitatively by a simple site binding model, providing binding parameters for the interaction between the amides and the nucleic acids both in the double- and the single-stranded conformation. According to these data the affinity to the single strands is higher than that to the double strands and increases in the series Phe less than Tyr less than Trp. The binding constants decrease with increasing salt concentration as expected for an interaction driven by electrostatic attraction. However, part of the interaction is also due to stacking between the aromatic amides and the nucleic acid bases. The present results indicate a direct correlation between the presence of aromatic amino acids at the binding site of helix destabilising proteins and the properties of simple derivatives of these amino acids. Furthermore the results suggest that very simple peptides containing aromatic amino acids served as a starting point for the evolution of helix destabilising proteins.     

The Evolutionary History of the Structure of 5S Ribosomal RNA

5S rRNA is the smallest nucleic acid component of the large ribosomal subunit, contributing to ribosomal assembly, stability, and function. Despite being a model for the study of RNA structure and RNA–protein interactions, the evolution of this universally conserved molecule remains unclear. Here, we explore the history of the three-domain structure of 5S rRNA using phylogenetic trees that are reconstructed directly from molecular structure. A total of 46 structural characters describing the geometry of 666 5S rRNAs were used to derive intrinsically rooted trees of molecules and molecular substructures. Trees of molecules revealed the tripartite nature of life. In these trees, superkingdom Archaea formed a paraphyletic basal group, while Bacteria and Eukarya were monophyletic and derived. Trees of molecular substructures supported an origin of the molecule in a segment that is homologous to helix I (α domain), its initial enhancement with helix III (β domain), and the early formation of the three-domain structure typical of modern 5S rRNA in Archaea. The delayed formation of the branched structure in Bacteria and Eukarya lends further support to the archaeal rooting of the tree of life. Remarkably, the evolution of molecular interactions between 5S rRNA and associated ribosomal proteins inferred from a census of domain structure in hundreds of genomes established a tight relationship between the age of 5S rRNA helices and the age of ribosomal proteins. Results suggest 5S rRNA originated relatively quickly but quite late in evolution, at a time when primordial metabolic enzymes and translation machinery were already in place. The molecule therefore represents a late evolutionary addition to the ribosomal ensemble that occurred prior to the early diversification of Archaea.     

Thermally driven fission of protocells

We propose a simple mechanism for the self-replication of protocells. Our main hypothesis is that the amphiphilic molecules composing the membrane bilayer are synthesized inside the protocell through exothermic chemical reactions. The slow increase of the inner temperature forces the hottest molecules to move from the inner leaflet to the outer leaflet of the bilayer. Because of this outward translocation flow, the outer leaflet grows faster than the inner leaflet. This differential growth increases the mean curvature and amplifies any local shrinking of the protocell until it splits in two. The proposed model, based on mere laws of physics, is a step in the study of the origin of life, as well as a clue for a better understanding of cell proliferation in cancer.     

Helix‐sheet packing in proteins

The three‐dimensional structure of a protein is organized around the packing of its secondary structure elements. Although much is known about the packing geometry observed between α‐helices and between β‐sheets, there has been little progress on characterizing helix–sheet interactions. We present an analysis of the conformation of αβ₂ motifs in proteins, corresponding to all occurrences of helices in contact with two strands that are hydrogen bonded. The geometry of the αβ₂ motif is characterized by the azimuthal angle θ between the helix axis and an average vector representing the two strands, the elevation angle ψ between the helix axis and the plane containing the two strands, and the distance D between the helix and the strands. We observe that the helix tends to align to the two strands, with a preference for an antiparallel orientation if the two strands are parallel; this preference is diminished for other topologies of the β‐sheet. Side‐chain packing at the interface between the helix and the strands is mostly hydrophobic, with a preference for aliphatic amino acids in the strand and aromatic amino acids in the helix. From the knowledge of the geometry and amino acid propensities of αβ₂ motifs in proteins, we have derived different statistical potentials that are shown to be efficient in picking native‐like conformations among a set of non‐native conformations in well‐known decoy datasets. The information on the geometry of αβ₂ motifs as well as the related statistical potentials have applications in the field of protein structure prediction. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

The "protocell": a mathematical model of self-maintenance

The concepts of self-generation, autonomous boundary and self-maintenance are explained briefly. The "protocell" is presented as a model of self-maintenance which is based on simple physical mechanisms of diffusion and reaction. The time evolution of the surface of the protocell is taken into account explicitly in the form of a Stefan condition giving rise to a non-linear feedback of the surface motion to the reaction and diffusion processes inside the protocell. The spatio-temporal dynamics are investigated, particularly in the neighbourhood of the stationary states, showing a self-maintaining behaviour under a certain range of nutritional conditions. Under another set of conditions we find an instability leading to a division process so that the population of protocells becomes self-maintaining instead of the single individual. The presented formulation of the protocell model is crucially improved compared with a previous version which required boundary conditions at infinity. The previous version was not strictly self-maintaining since dynamics outside the cell were essential for its behaviour.     

Early Stages of the Evolution of Life: a Cybernetic Approach

Early stages of the evolution of life are considered in terms of control theory. A model is proposed for the transport of substances in a protocell possessing the property of robustness with regard to changes in the environmental concentration of a substance.     

A Fission-Fusion Origin for Life

To develop a comprehensive cells-first approach to the origin of life, we propose that protocells form spontaneously and that the fission and fusion of these protocells drives the dynamics of their evolution. The fitness criterion for this evolution is taken to be the the stability (conservation) of domains in the protocellular membrane as determined by non-covalent molecular associations between the amphiphiles of the membrane and a subset of the macromolecules in the protocell. In the presence of a source of free energy the macromolecular content of the protocell (co-)evolves as the result of (domain-dependent) membrane-catalysed polymerisation of the prebiotic constituents delivered to the protocell by fusion. The metabolism of the cell therefore (co-)evolves on a rugged fitness landscape. We indicate how domain evolution with the same fitness criterion can potentially give rise to coding. Membrane domains may therefore provide the link between protocells and the RNA/DNA-world.     

Generic Darwinian selection in catalytic protocell assemblies

To satisfy the minimal requirements for life, an information carrying molecular structure must be able to convert resources into building blocks and also be able to adapt to or modify its environment to enhance its own proliferation. Furthermore, new copies of itself must have variable fitness such that evolution is possible. In practical terms, a minimal protocell should be characterized by a strong coupling between its metabolism and genetic subsystem, which is made possible by the container. There is still no general agreement on how such a complex system might have been naturally selected for in a prebiotic environment. However, the historical details are not important for our investigations as they are related to assembling and evolution of protocells in the laboratory. Here, we study three different minimal protocell models of increasing complexity, all of them incorporating the coupling between a 'genetic template', a container and, eventually, a toy metabolism. We show that for any local growth law associated with template self-replication, the overall temporal evolution of all protocell's components follows an exponential growth (efficient or uninhibited autocatalysis). Thus, such a system attains exponential growth through coordinated catalytic growth of its component subsystems, independent of the replication efficiency of the involved subsystems. As exponential growth implies the survival of the fittest in a competitive environment, these results suggest that protocell assemblies could be efficient vehicles in terms of evolving through Darwinian selection.     

Quantum processes in evolution of regulation of living system (mathematical modelling)

We have developed an imitation model of the appearance of regulation of physiological functions of protocell at the initial stages of evolution of living system. It is based on suggestion of the appearance of signal function in spontaneously formed products of partial hydrolysis of the protocell polypeptides, based on which there appear the regulatory molecules--quanta of regulation. For construction of the model, the mathematical apparatus of final automats and of genetic algorithm is used. The model has demonstrated the positive role of involvement of regulatory peptides in the system of regulation of protocell functions to provide its viability under the changing envelopment conditions.     

Pause point spectra in DNA constant-force unzipping

Under constant applied force, the separation of double-stranded DNA into two single strands is known to proceed through a series of pauses and jumps. Given experimental traces of constant-force unzipping, we present a method whereby the locations of pause points can be extracted in the form of a pause point spectrum. A simple theoretical model of DNA constant-force unzipping is presented, which generates theoretical pause point spectra through Monte Carlo simulation of the unzipping process. The locations of peaks in the experimental and theoretical pause point spectra are found to be nearly coincident below 6000 basepairs for unzipping the bacteriophage lambda-genome. The model only requires the sequence, temperature, and a set of empirical basepair binding and stacking energy parameters, and the good agreement with experiment suggests that pause point locations are primarily determined by the DNA sequence. The model is also used to predict pause point spectra for the bacteriophage phi X174 genome. The algorithm for extracting the pause point spectrum might also be useful for studying related systems which exhibit pausing behavior such as molecular motors.     

Teaching the origin of the first living systems

The most fundamental of questions in biology, namely that of the origin of living systems, is being lost to teaching and a new technique to rekindle interest in it must be found. This paper presents a novel idea of teaching a scientific concept using a poem, which describes the major perspectives on the origins of living systems, as the medium of instruction. All of the major schools of thought — chemical evolution, DNA vs. RNA, protocell formation, coacervates, panspermia and special creation — are discussed. The aim of the paper is not to be a definitive review on the origin of living systems, but rather to be a focal point on which to hinge further discussion.     

Comments on selected aspects of nucleic acid electrostatics

Recent experimental, theoretical, and computational developments in the field of nucleic acid electrostatics have brought interesting concepts to the fore. The phosphate charge on the double helix apparently influences its structure. When the charge is neutralized asymmetrically, the resulting force imbalance drives bending toward the neutralized side. When the charge is uniformly neutralized, the force imbalance acts to buckle the helical axis, resulting in a compact tertiary conformation. Sharing of condensed counterions by single strands is a stabilizing factor for formation of the double helix. Sharing of condensed counterions by two double helices causes clustering of DNA and may be a factor in RNA folding. Support for these statements is reviewed.     

Quantum processes in evolution of regulation of living systems (mathematical modeling)

We have developed an imitation model of the appearance of regulation of physiological functions of protocell at initial stages of evolution of living system. It is based on suggestion of the appearance of signal function in spontaneously formed products of partial hydrolysis of the protocell polypeptides, based on which there appear the regulatory molecules—quanta of regulation. For construction of the model, the mathematical apparatus of final automats and of genetic algorithm is used. The model has demonstrated the positive role of involvement of regulatory peptides in the system of regulation of protocell functions to provide its viability under the changing envelopment conditions.     

PNA technology

Peptide nucleic acids (PNA) are deoxyribonucleic acid (DNA) mimics with a pseudopeptide backbone. PNA is an extremely good structural mimic of DNA (or of ribonucleic acid [RNA]), and PNA oligomers are able to form very stable duplex structures with Watson-Crick complementary DNA and RNA (or PNA) oligomers, and they can also bind to targets in duplex DNA by helix invasion. Therefore, these molecules are of interest in many areas of chemistry, biology, and medicine, including drug discovery, genetic diagnostics, molecular recognition, and the origin of life. Recent progress in studies of PNA properties and applications is reviewed.     

Analysis of superhelical structures of nucleic acid-lipid conjugates by image processing

Nucleoside-phospholipid conjugates containing a nucleotidyl residue and two long alkyl chains have been synthesized and their self-organization and morphology have been investigated. In particular, 5'-phosphatidylcytidine spontaneously assembled to form linear and circular strands. Image processing analysis of the electron micrograph of the strands confirmed that they are indeed double helix reminiscent of the double-helical structure of nucleic acids. The linear and circular strands from 5'-phosphatidylcytidine had grooves of approximately 100 A in diameter and right-handed helical pitch of approximately 240 A.