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CHEMICAL AND BIOLOGICAL DISCOVERY DRIVEN BY EVOLUTION

Professor David R. Liu
Department of Chemistry and Chemical Biology, Harvard University
Howard Hughes Medical Institute

drliu@fas.harvard.edu http://evolve.harvard.edu

Overview

Our laboratory develops and applies new approaches to chemical and biological discovery that are driven by the principles underlying biological evolution. Over the past five years we initiated two new lines of research. The first aims to precisely manipulate information flow in mammalian cells through the use of synthetic regulatory elements (SREs), proteins and nucleic acids that modify genes and gene products with tailor-made specificities. Achieving this ambitious goal requires addressing two longstanding challenges in the molecular life sciences: (i) the development of a system for the continuous directed evolution of proteins and nucleic acids, and (ii) the development of a general platform for the delivery of macromolecules into mammalian cells in vitro and in vivo. Our research efforts over the past five years resulted in new solutions to both challenges. The second new line of research seeks to discover cellular nucleic acids with novel structures and functions, and resulted in the discovery of cofactor-linked RNAs, geranylated RNAs, and a new DNA nucleotide. Finally, we continue to pioneer the development and application of DNA-templated synthesis (DTS), resulting in the discovery of several new small-molecule inhibitors of disease-associated proteins and three new chemical reactions over the past five years. A sample of research progress (not comprehensive) over the past five years in each of these three areas is summarized below.

Introduction to Area 1: The Creation and Use of Synthetic Regulatory Elements in Mammalian Cells

Our emerging understanding of living systems has made increasingly realistic the possibility of manipulating cellular information flow by precisely altering genome or proteome structure, engineering new conditions under which genes are expressed, or creating new information-processing circuits that interface exogenous molecules with genes. The purposeful manipulation of information flow in living systems has been a longstanding goal of the molecular life sciences, and if applied to humans could lead to breakthrough diagnostics and therapeutics that respond in sophisticated ways to disease. A number of daunting challenges must be overcome before this vision can be realized. Perhaps the two most significant of these challenges are how to create with a practical efficiency and success rate synthetic regulatory elements (SREs) that alter genomes, transcriptomes, or proteomes; and how to deliver macromolecules into mammalian cells in vitro and in vivo in ways that preserve their activities and minimize the risk of undesired side effects.

Area 1: A System for the Continuous Directed Evolution of Proteins and Nucleic Acids

Macromolecule engineering and directed evolution efforts in our laboratory and in other groups have resulted in a number of relevant successes including recombinases that target pathogen genomes,1 synthetic riboswitches that respond to small molecules,2-7 ligand-dependent inteins that render protein activities dependent on a cell-permeable small molecule,8-12 and nucleases with tailor-made DNA specificities.13,14 Such examples also highlight the difficulty of generating these macromolecules, as each required several person-years of effort. To realize a vision in which arbitrary genes, transcripts, or proteins are manipulated at will in mammalian cells, much more rapid and effective approaches to generating SREs must be developed.

Conventional directed evolution involves discrete cycles of mutagenesis, transformation or in vitro expression, screening or selection, and gene harvesting.15,16 Although successful evolution is strongly dependent on the total number of rounds performed,17 the labor- and time-intensive nature of this process limit laboratory evolution efforts to a modest number of rounds. Continuous directed evolution, in which mutation, transformation, selection, and replication takes place constantly, without requiring researcher intervention, has the potential to dramatically enhance the speed and effectiveness of laboratory evolution 18,19 but has only been previously implemented in one landmark example by Joyce and co-workers to evolve ribozymes20,21 using a method that cannot be easily adapted to other biomolecules.

We harnessed the life cycle of filamentous bacteriophage to develop a system for the continuous directed evolution of proteins and nucleic acids.22 In phage-assisted continuous evolution (PACE), E.coli host cells continuously flow through a "lagoon" containing an actively evolving population of phage DNA vectors encoding the genes of interest.22 During PACE the desired activity is linked to the production of a protein required for the production of infectious progeny phage containing the evolving genes. Due to the speed of the phage life cycle,23 PACE can mediate ~40 complete rounds of evolution per day, a 100- fold increase in speed over conventional methods.22 Importantly, PACE requires no intervention during evolution and obviates the need to create DNA libraries, transform cells, or extract genes during each round.

We used PACE to continuously evolve three new activities in a protein enzyme. Although the starting enzyme exhibits undetectable levels of activity in two of these cases, enzymes with at least wild- type-levels of each target activity emerged in only 1-8 days of PACE, corresponding to 45-200 rounds of evolution.22 By accelerating directed evolution ~100-fold, PACE may provide solutions to otherwise intractable directed evolution problems and address novel questions about molecular evolution.

Area 1: Supercharged Proteins as a General Macromolecule Delivery Platform

The development of a general platform to deliver macromolecules into mammalian cells in vitro and in vivo would represent a key step towards the application of SREs in living systems, and would also address the major challenge facing the use of proteins and nucleic acids as intracellular probes and therapeutic agents.

We discovered that "supercharging" proteins--replacing their surface-exposed residues with Lys/Arg, or with Glu/Asp--can impart immunity to aggregation without abolishing their native fold or function.24 We also found that superpositively charged proteins such as +36 GFP very potently enter mammalian cells, including four cell lines resistant to traditional transfection methods.25 Supercharged GFPs also delivered siRNA and plasmid DNA potently and without cytotoxicity in these cells, resulting in siRNA-based gene silencing or plasmid- based gene expression.25 +36 GFP also rapidly delivered proteins including active, non-endosomal ubiquitin and Cre recombinase into all five cell lines tested with up to 100- fold greater potency than the currently used protein transduction domains (PTDs) Tat, Arg10, and penetratin.26 We have also gained new insights into the mechanism of cell entry and the mechanistic basis of the macromolecule delivery potency of supercharged proteins.

We recently discovered a substantial class of natural proteins with similar cell penetration and macromolecule delivery properties both in vitro and in vivo. Members of this class of proteins may represent a more immunologically tolerated platform for in vivo protein delivery than other macromolecular agents. Moreover, the ability of many of these proteins to penetrate cells has not been previously reported, and our findings raise the possibility that this substantial class of natural proteins may play undiscovered biological roles that arise from their previously unknown ability to penetrate cells.

Introduction to Area 2: A Broad Search for New Structures and Functions in Cellular Nucleic Acids

Over the past few decades, RNA has emerged as much more than an intermediary in biology’s central dogma, and many RNAs are now known to play a wide range of catalytic, regulatory, or defensive roles in the cell.28-37 In contrast with its broad functional diversity, the known chemical diversity of biological RNA has remained limited primarily to canonical ribonucleotides, 3’-aminoacylated tRNAs,38 modified nucleobases,38 and 5’-capped mRNAs.40-42 This disparity between RNA’s known functional and chemical diversity, coupled with the powerful properties of synthetic small molecule-nucleic acid conjugates (see Area 3 below), led us to speculate that small molecule-RNA conjugates beyond those previously described may exist in cells as evolutionary fossils or as RNAs with novel functions enabled by their modifications.

Area 2: Discovery of Cellular CoA-RNA, NAD-RNA, Geranylated RNA, and a New DNA Nucleotide

We developed and implemented three increasingly general chemical screens to discover cellular small molecule-RNA conjugates.43,44 In contrast with previous efforts, our methods are not dependent on any specific type of small- molecule structure or any particular biological function of the conjugate. Once the structures of the small molecules are solved, we isolate the attached RNA(s) by using chemoselective capture or by testing specific RNAs that we hypothesize may be linked to the small molecules.

Using this approach, we identified coenzyme A (CoA) and several CoA thioesters (acetyl, malonyl, and methyl malonyl-CoA) as covalent conjugates to cellular RNA in E. coli and S. venezuelae.44 Our experiments indicate that the CoA -derived RNA(s) are under ~200 nucleotides in length, contain CoA at their 5’ ends, and are probably not generated by aberrant transcriptional initiation.44 We are currently using thiol capture and high-throughput sequencing in an effort to identify these RNAs as the next step towards understanding their potential biological roles.

Our methods have also revealed NAD-linked RNA43 which we discovered in E. coli, S. venezuelae, B. subtilis,E. aerogenes, V. fischeri, and bovine RNA. NAD- RNA is surprisingly abundant in cells at a level of ~3,300 copies per E. coli cell, comparable to the total number of mRNA molecules in an E. coli cell.43 The most recent small molecule-RNA conjugates discovered with our approach are two lipid-like nucleotide derivatives, which we isolated from a variety of organisms. These small molecule-RNA conjugates include the first cofactor-linked RNA isolated from cells. More generally, our findings reveal that the chemical diversity of biological RNA in modern cells is greater than previously understood.

In collaboration with Professor Anjana Rao (Harvard Medical School), we elucidated the structure of a new DNA nucleotide generated in mammalian cells upon overexpression of TET1, an enzyme that Rao and coworkers hypothesized may be a methylcytosine-modifying enzyme.45 Using the comparative mass spectrometry screening methods that we developed to study RNA, we discovered that this novel DNA nucleotide is 5-hydroxymethylcytosine (hmC), suggesting that TET1 may play a role in epigenetic regulation of the mammalian genome by mediating the oxidation of methylcytosine to hmC, a potential key step in the demethylation of 5-methylcytosine.

Area 3 Introduction: Components of Evolution Applied to Synthetic Molecules Via DNA-Templated Synthesis

Several years ago our laboratory developed a new approach to the synthesis and discovery of small molecules that combines powerful aspects of natural biosynthesis and evolution with the flexibility of synthetic organic chemistry. We discovered that DNA hybridization induces the rapid, general, and sequence-programmed reaction of DNA-linked substrates, enabling DNA-templated organic synthesis (DTS).46-48,42 By translating DNA sequences into synthetic molecules, DTS enables selection, amplification, and mutation to be applied to molecules that can only be accessed through chemical synthesis. Over the past five years we performed the first large-scale, systematic application of DTS and in vitro selection, resulting in the discovery of many bioactive synthetic molecules and three new chemical reactions.

Area 3: Discovery of Bioactive Macrocycles from DNA-Templated Synthesis and Selection

Integrating many of our recent developments in DTS,49-54 we translated a library of DNA templates in one solution into a corresponding library of ~13,824 DNA-linked macrocycles.55 We subjected this macrocycle library to highly efficient in vitro selections51 for binding to several dozen proteins of current biomedical interest. The selection results identified macrocycles that inhibit kinases implicated in human cancers56 and a metalloprotease implicated in diabetes. Two families of macrocyclic Src inhibitors (IC50 < 1 µM) exhibit remarkable selectivity for Src over other human kinases.56 We improved the potency of this class of Src inhibitors to IC50 <= ~1 nM and confirmed that the resulting compounds can inhibit Src in human cells. These results established the use of DNA-templated synthesis and in vitro selection to discover small molecules that modulate biological activities, an approach that has since been industrialized. In a collaborative effort with Professor Markus Seeliger (SUNY Stonybrook), the X-ray structure of Src kinase complexed with one of these macrocyclic inhibitors was solved, revealing detailed molecular insights into the macrocycles binding, inhibition, and unusual specificity.

Area 3: DNA-Encoded Reaction Discovery

In the past five years we developed a second-generation DNA-encoded reaction discovery system and used it to discover two new reactions, validating a non-hypothesis-driven approach to reaction discovery that has now been adopted by some synthetic chemistry groups.57,58 The first reaction is a mild Au(III)- or triflic acid-catalyzed hydroarylation of olefins with indoles.57 The second is a visible light-triggered, Ru(II)-catalyzed reduction of azides to amines that has a remarkable functional group tolerance.58 Indeed, this azide reduction reaction can be performed on nucleic acid and carbohydrate substrates, and in the presence of protein enzymes, without modifying any groups present on these macromolecules beyond the azide.58 This reaction therefore provides a means of photouncaging a widely used chemical handle in a manner that is biomolecule-compatible.

Area 3: RDPCR and IDPCR

We developed two replacements for conventional in vitro selections, reactivity-dependent PCR (RDPCR)59 and interaction-dependent PCR (IDPCR),60 that have transformed the way we use DNA-linked molecules. RDPCR directly links bond formation with the selective PCR of DNA encoding reactive library members without the need for substrate immobilization or solid-phase capture and elution steps.59 Reaction discovery using RDPCR resulted in a new Pd(II)-catalyzed allene-azide coupling reaction that generates azidolactones, useful intermediates that would otherwise be more challenging to synthesize. Likewise, IDPCR replaces solid-phase binding selections and enables for the first time productive ligand-target combinations to be identified in a single experiment from combined libraries of small-molecule ligands and targets.60

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