Contemporary phospholipid based cell membranes are formidable barriers to the uptake of polar and charged molecules ranging from metal ions to complex nutrients. Modern cells therefore require sophisticated protein channels and pumps to mediate the exchange of molecules with their environment. The strong barrier function of membranes has made it difficult to understand the origin of cellular life and has been thought to preclude a heterotrophic lifestyle for primitive cells. Although nucleotides can cross DMPC membranes through defects formed at the gel to liquid transition temperature 1, 2 , phospholipid membranes lack the dynamic properties required for membrane growth. Fatty acids and their corresponding alcohols and glycerol monoesters are attractive candidates for the components of protocell membranes because they are simple amphiphiles that form bilayer membrane vesicles 3-5 that retain encapsulated oligonucleotides 3,6 and are capable of growth and division 7-9 . Here we show that such membranes allow the passage of charged molecules such as nucleotides, so that activated nucleotides added to the outside of a model protocell (Fig. 1) spontaneously cross the membrane and take part in efficient template copying in the protocell interior. The permeability properties of prebiotically plausible membranes suggest that primitive protocells could have acquired complex nutrients from their environment in the absence of any macromolecular transport machinery, i.e. could have been obligate heterotrophs.Previous observations of slow permeation of UMP across fatty acid based membranes 6 stimulated us to explore the structural factors that control the permeability of these membranes. We examined membrane compositions with varied surface charge density, fluidity, and stability of regions of high local curvature. We began by studying the permeability of ribose, because this sugar is a key building block of the nucleic acid RNA, and because sugar permeability is conveniently measured with a real-time fluorescence readout of vesicle volume following solute addition 10, 11 . We used pure myristoleic acid (C14:1 fatty acid, myristoleate in its ionized form) as a reference composition, because this compound generates robust vesicles that are more permeable to solutes than the more common longer chain oleic acid. Both myristoleyl alcohol (MA-OH) and the glycerol monoester of myristoleic acid (monomyristolein, GMM) stabilize myristoleate vesicles to the disruptive effects of divalent cations 3,6 . Addition of these amphiphiles should decrease the surface charge density of myristoleate vesicles, while myristoleyl phosphate (MP) should increase the surface charge

The development of a sequence-general nucleic acid copying system is an essential step in the assembly of a synthetic protocell, an autonomously replicating spatially localized chemical system capable of spontaneous Darwinian evolution. Previously described nonenzymatic template-copying experiments have validated the concept of nonenzymatic replication, but have not yet achieved robust, sequence-general polynucleotide replication. The 5′-phosphorimidazolides of the 2′-amino-2′,3′-dideoxyribonucleotides are attractive as potential monomers for such a system because they polymerize by forming 2′→5′ linkages, which are favored in nonenzymatic polymerization reactions using similarly activated ribonucleotides on RNA templates. Furthermore, the 5′-activated 2′-amino nucleotides do not cyclize. We recently described the rapid and efficient nonenzymatic copying of a DNA homopolymer template (dC15) encapsulated within fatty acid vesicles using 2′-amino-2′,3′-dideoxyguanosine−5′-phosphorimidazolide as the activated monomer. However, to realize a true Darwinian system, the template-copying chemistry must be able to copy most sequences and their complements to allow for the transmission of information from generation to generation. Here, we describe the copying of a series of nucleic acid templates using 2′-amino-2′,3′-dideoxynucleotide−5′-phosphorimidazolides. Polymerization reactions proceed rapidly to completion on short homopolymer RNA and LNA templates, which favor an A-type duplex geometry. We show that more efficiently copied sequences are generated by replacing the adenine nucleobase with diaminopurine, and uracil with C5-(1-propynyl)uracil. Finally, we explore the copying of longer, mixed-sequence RNA templates to assess the sequence-general copying ability of 2′-amino-2′,3′-dideoxynucleoside−5′-phosphorimidazolides. Our results are a significant step forward in the realization of a self-replicating genetic polymer compatible with protocell template copying and suggest that N2′→P5′-phosphoramidate DNA may have the potential to function as a self-replicating system.