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The key concepts of genetics are phenotype and genotype: genotype is generally embodied in a nucleic acid sequence, and phenotype represents some observable property of an organism. Moving these ideas from the organismal to the molecular level leads naturally to the idea of creating and manipulating genetic variation in molecules, entirely in vitro. The discovery of catalytic RNAs in the early 1980s demonstrated that RNA could manifest a range of interesting phenotypes. Simultaneously, the invention of the polymerase chain reaction made it easy to replicate almost any nucleic acid sequence in vitro. Together these advances inspired the development of practical ways of using in vitro selection and directed evolution to search sequence space, the ensemble of all possible sequences, for new functional RNA, DNA, and protein sequences.
The simplest interesting phenotypes of RNAs and DNAs are binding properties. In vitro selection for sequences that fold up into specific three-dimensional structures that contain highly specific binding sites has been used to isolate many nucleic acids, called aptamers, that bind a wide range of small biomolecules, including nucleotides, amino acids, antibiotics, and cofactors. We have found that these nucleic acid molecules can be selected successfully for catalysis as well.
Application of the principles of in vitro selection and directed evolution to peptides and proteins is a powerful tool for investigating protein function and structure and for obtaining insight into the pathways by which enzymes evolve in nature. Our approach has been to generate stable, covalent RNA-protein fusions in a completely in vitro system. We do this by covalently linking puromycin, an antibiotic that mimics an aminoacylated tRNA, to the 3’ end of a synthetic mRNA through a DNA linker. A ribosome begins translation of such a template as usual, generating a peptide as it transits the open reading frame. When the ribosome reaches the end of the open reading frame and hits the DNA linker it stalls, allowing the nearby puromycin to enter the A site of the ribosome and accept the nascent peptide chain. The resulting RNA-peptide fusions can be formed efficiently from mRNAs encoding small peptides or large proteins.
We have prepared libraries of fusions encoding random peptides and are preparing to begin evolving new binding domains and enzymes. An exciting future application will be the ability to conduct side-by-side comparisons of RNA and protein evolution. (This work was supported in part by grants from the National Institutes of Health.)
Dr. Szostak is an Investigator of the Howard Hughes Medical Institute, Professor of Genetics at Harvard Medical School, Professor of Chemistry and Chemical Biology (Faculty of Arts and Sciences) at Harvard University and the Alex Rich Distinguished Investigator in the Department of Molecular Biology at the Massachusetts General Hospital.
His current research interests are in the laboratory synthesis of self-replicating systems and the origin of life. He and his colleagues have developed in vitro selection as a tool for the isolation of rare functional RNA, DNA and protein molecules from large pools of random sequences. His laboratory has used in vitro selection and directed evolution to isolate and characterize numerous nucleic acid sequences with specific ligand binding and catalytic properties.
For this work, Dr. Szostak was awarded, along with Dr. Gerald Joyce, the 1994 National Academy of Sciences Award in Molecular Biology and the 1997 Sigrist Prize from the University of Bern. Dr. Szostak is a member of the National Academy of Sciences, and a Fellow of the New York Academy of Sciences and the American Academy of Arts and Sciences. In 2000, Dr. Szostak was awarded the Medal of the Genetics Society of America. Dr. Szostak has been awarded the 2009 Nobel Prize in Physiology or Medicine for his original contributions to our understanding of the processes of life and of disease.
The key tool for the selection of protein sequences with novel binding or catalytic activity is mRNA Display (FIGURE and description). The mRNA-display approach uses the peptidyl-accepting antibiotic puromycin, chemically appended to the 3’ of the mRNA, to provide a covalent link between the nascent peptide and its coding mRNA prior to release from the ribosome. This physical linkage between the ribosomally synthesized protein (phenotype) and the mRNA that codes for its synthesis (genotype) allows for the simultaneous isolation of the desired functional protein and the encoding mRNA.
While mRNA display has been used extensively by our laboratory and others in the selection of proteins with novel sequences comprised of the 20 naturally-occurring amino acids, we have greatly expanded its utility by establishing conditions for the incorporation non-natural amino acids. Using a fully-defined in vitro translation system comprised of highly purified E. coli ribosomes, tRNAs, and recombinant translation factors and amino acid tRNA synthetases, and omitting naturally-occurring amino acids, we have been able to charge individual tRNAs with a wide variety of non-natural amino acids. These charged tRNAs can participate in mRNA-directed, ribosome-mediated peptide-bond synthesis. This system is fully compatible with the synthesis of puromycin-mediated molecular fusions between the unnatural peptide and its mRNA for the in vitro selection of drug-like molecules by mRNA-display.
Non-natural amino acids under evaluation include D-amino acids, beta-amino acids, N-methyl amino acids, and amino acids with modified, novel, or chemically-reactive side chains. We predict that peptides synthesized by this approach will greatly expand the chemical diversity of peptide libraries and further allow the synthesis of peptides with desirable “drug-like” properties such as protease resistance, chemical stability, cyclic structures, and enhanced binding capabilities with in vivo targets.
In Vitro Selection
The Szostak Laboratory has pioneered the development of in vitro techniques for selecting novel RNA and protein molecules with high-affinity binding or enzymatic activity from highly diverse, random polymer pools. RNA or protein “aptamers” exhibiting high affinity and specific binding to a variety of small molecule targets have been isolated, including RNA molecules that selectively bind ATP, GTP, biotin, riboflavin, nicotinamide, and vitamin B12, as well as protein molecules that bind ATP and streptavidin. A number of in vitro-selected catalytic RNA molecules, or ribozymes, have been isolated and characterized, including ribozymes with ligase, kinase and acyl transferase activities, and efforts are underway to isolate novel proteins with catalytic functions.
We are also interested in exploring the capabilities of polymers with nucleic-acid like properties that have more plausible routes to prebiotic synthesis than either RNA or DNA. Threose Nucleic Acid (TNA) has been suggested as a candidate because of its ability to adopt A-form geometry and base-pair with RNA and DNA. To explore the functionality of TNA we have developed a system for performing in vitro selections for TNA sequences with desirable structural and functional properties. In this approach, TNA is enzymatically transcribed from a single-stranded portion of DNA using a haipin primer. A round of DNA synthesis is then used to displace a single-stranded region of TNA which may be subjected to in vitro selection. This design maintains a physical link between the TNA sequence and the complementary DNA sequence (see figure above), which greatly facilitates amplification for subsequent rounds of selection.
The work of our laboratory and by others has clearly demonstrated that in vitro selection techniques can be used to isolate RNA and protein sequences that bind to essentially any molecular target or catalyze any chemical reaction, providing powerful tools for drug discovery.
The Origins of Life
Proposed pathway for cell synthesis (click to enlarge)
It is generally accepted that life in its present form — with proteins performing most enzymatic functions and nucleic acids comprising the genetic material — was preceded by a period in which nucleic acid, either RNA or a closely related polymer, filled both roles.
The laboratory is utilizing a diverse array of molecular biological and chemical tools to create model systems for the study of the origin of life. The main goals are:
- to establish a system for the replication of nucleic acids that does not rely on proteins
- to establish physical and chemical methods for the growth and division of fatty acid vesicles
- to encapsulate a replicating nucleic acid system in fatty acid vesicles to create protocells
- to subject protocells to Darwinian selection in order to understand the evolution of complex metabolism
The Szostak Lab has participated in the development of a web site exploring the origins of life in parallel with its own research goals on the origins and development of the first living cells on Earth. “Exploring Life’s Origins” describes current research on the beginnings of life to a broad nontechnical audience using three-dimensional molecular visualizations. The website is part of a multimedia exhibit on the origins of life at the Boston Museum of Science.
Click here to see the web presentation created by Janet Iwasa of the Szostak Lab.
Self-Replicating Nucleic Acids
A prerequisite for constructing a protocell is supplying a mechanism for replicating the nucleic acids that fill structural, functional or informational roles. Two approaches under active investigation in our laboratory are the in vitro selection of ribozyme polymerases (replicases) and the non-enzymatic replication of nucleic acids using chemically-activated nucleotides. In both cases, the goal is a high-fidelity system for replicating essentially any nucleotide sequence.
Ribozyme polymerases are being selected from fully random or partially structured libraries of RNA or by modification of natural ribozymes with reversible phosphodiesterase bond cleaving activity.
Non-enzymatic replication utilizes chemically-activated monomers or condensing agents to effect polymerization of monomers on template sequences. Chemical activation requires substituting a preferred leaving group in place of one of the hydroxyl groups of the 5´phosphate in a nucleoside monophosphate. Although this chemistry is well established in the literature, it has been limited by the slow reaction rates of naturally-occurring 3´OH-terminated nucleotides. We have recently found that substitution of the 3´OH with an amino group dramatically increases the reaction rate with activated monomers. The product, phosphoramidate DNA, is a well-known polymer with properties similar to naturally-occurring DNA and RNA. We are currently synthesizing a variety of chemically-activated nucleotides with 3´NH2 substitutions and are evaluating the kinetics and fidelity of non-enzymatic polymerization.
Ultimately, we hope to establish a robust and efficient system for template-directed synthesis of NP DNA that can be incorporated into fatty acid vesicles. Based on the known properties of NP DNA, we expect that it can adopt the types of compact structures characteristic of RNA aptamers and ribozymes and will be a rich source of functionally active sequences. Combined with a membrane component for compartmentalization and a system for non-enzymatic replication, we believe that NP DNA may be able to provide the functional and information-carrying needs of a simple cell. To reach this goal we are optimizing the structures of 3´NH2 substituted nucleotides, establishing methods for the in situ activation of nucleotides using condensing agents, evolving protein enzymes capable of synthesizing NP DNA from DNA templates and vice versa to facilitate in vitro selections, and gaining a thorough biochemical and biophysical understanding of NP DNA through experimentation.
Primitive cells, lacking the complex biomachinery present in modern cells, would have had to rely on the self-organizing properties of their components and on interactions with their environment to achieve basic cellular functions such as growth and division.
Proposed pathway for cell synthesis (click to enlarge)
Many bilayer-membrane vesicles can exhibit complex morphological changes such as growth, fusion, fission, budding, internal vesicle assembly and vesicle-surface interactions. The rich dynamic properties of these vesicles provide interesting models of how primitive cellular replication might have occurred in response to purely physical and chemical forces.
We have engineered a system of vesicle replication with discrete growth and division steps. When micelles comprised of myristoleate were added to myristoleic acid/myristoleate vesicles, we observe vesicle growth as well as new vesicle formation. By controlling the rate of addition of micelles to eliminate high concentrations that favor their self-association, vesicle growth can be promoted at the expense of new vesicle formation.
Osmotic swelling of vesicles produces a high energy state as compared to relaxed (isotonic) vesicles, and membrane components may be transferred from relaxed, low energy vesicles to swollen, high energy vesicles, minimizing the overall energy of the mixture and resulting in the growth of the swollen vesicles. In one example of this phenomenon, RNA encapsulated in fatty acid vesicles is used to exert an osmotic pressure on the vesicle membrane that drives the uptake of additional membrane components, leading to membrane growth at the expense of relaxed vesicles, which shrink. This observation supports a model in which more efficient RNA replication could result to more rapid vesicle growth, leading to the emergence of Darwinian evolution in primitive cells.
We have also shown that mineral surfaces can accelerate the assembly of vesicles from fatty acid micelles. A dispersion of montmorillonite clay in buffer accelerates the assembly of vesicles from fatty acid micelles. When the vesicles form, some of the clay becomes encapsulated in large vesicles, which in turn become packed full of smaller vesicles. In addition, RNA adsorbed to clay can be encapsulated within vesicles. Since oligonucleotides can be synthesized on clay surfaces, clay-catalyzed vesicle assembly provides a direct path for the incorporation of nucleic acids into vesicles. The formation, growth, and division of the earliest cells may have occurred in response to similar interactions with mineral particles and inputs of material and energy.
Work in our laboratory has recently demonstrated that a pH gradient can form spontaneously across the membrane of growing vesicles. As models of primitive cellular life, replicating vesicles must also provide a means of harvesting energy and small molecules to fuel a primitive metabolism. The generation of pH gradients due to vesicle growth suggests that it may be possible to capture some of the energy released during growth in a form that could be used for other processes, such as substrate uptake.
Division of vesicles can be achieved by extruding the grown vesicles through small pores, so that the extruded vesicles have the same diameter as the initial population before vesicle growth. By monitoring a fluorescent dye encapsulated within the initial vesicles, we have shown that the division process occurs with very little loss or dilution of the vesicle contents over multiple cycles of growth and extrusion. This simple system serves as a proof of principle that primitive cellular replication could have occurred through purely physico-chemical forces, such as fluid flowing through porous rocks near hydrothermal vents.
Click here for a video of vesicle extrusion [.avi format, approx. 7MB] Left-click to play the video, or right-click and choose “Save Target As” to download a copy to your PC.
Developing Vesicles to Support Ribozyme Function
Our efforts to build a simple cell comprised of a membrane and a nucleic acid-based metabolism begin with establishing conditions that allow ribozymes to function within vesicles. Using a mixture of myristoleic acid and its glycerol monoester we have constructed vesicles that were Mg2+-tolerant. Membranes made from these simple amphiphiles can form vesicles that are stable enough to retain encapsulated RNAs in the presence of divalent cations, yet dynamic enough to grow spontaneously and allow the passage of Mg2+ and mononucleotides without specific macromolecular transporters.
A hammerhead ribozyme was incorporated into vesicles produced in this manner. The addition of external Mg2+ led to the activation and self-cleavage of the ribozyme molecules.
This combination of stability and dynamics is critical for building model protocells in the laboratory and may have been important for early cellular evolution.
The Chemical Bonding Center at MGH – Describes a new collaborative initiative for the development of artificial chemical systems
“NIH/National Library of Medical Porta”l http://www.ncbi.nlm.nih.gov
HHMI/Keck Biopolymer Facility at Yale – Specialty oligonucleotide synthesis
The RNA World at IMB, Jena – Maintains large number of links to sites about RNA
Vienna RNA Package – RNA folding and sequence design software