A fundamental problem in research on the origin of life is the process by which polymers capable of catalysis and replication were produced on the early Earth. Here we show that RNA-like polymers can be synthesized non-enzymatically from mononucleotides in lipid environments. The RNA-like polymers were initially identified by nanopore analysis, a technique with single molecule sensitivity. To our knowledge, this is the first such application of a nanopore instrument to detect RNA synthesis under simulated prebiotic conditions. The synthesis of the RNA-like polymers was confirmed by standard methods of enzymatic end labeling followed by gel electrophoresis. Chemical activation of the mononucleotides is not required. Instead, synthesis of phosphodiester bonds is driven by the chemical potential of fluctuating anhydrous and hydrated conditions, with heat providing activation energy during dehydration. In the final hydration step, the RNA-like polymer is encapsulated within lipid vesicles. This process provides a laboratory model of an early stage of evolution toward an RNA World.

Nanoscale pores have potential to be used as biosensors and are an established tool for analysing the structure and composition of single DNA or RNA molecules 1-3 . Recently, nanopores have been used to measure the binding of enzymes to their DNA substrates 4,5 . In this technique, a polynucleotide bound to an enzyme is drawn into the nanopore by an applied voltage. The force exerted on the charged backbone of the polynucleotide by the electric field is used to examine the enzyme-polynucleotide interactions. Here we show that a nanopore sensor can accurately identify DNA templates bound in the catalytic site of individual DNA polymerase molecules. Discrimination among unbound DNA, binary DNA/polymerase complexes, and ternary DNA/polymerase/ deoxynucleotide triphosphate complexes was achieved in real time using finite state machine logic. This technique is applicable to numerous enzymes that bind or modify DNA or RNA including exonucleases, kinases and other polymerases.We describe a nanopore device that monitors ionic current through a single protein pore inserted in a lipid bilayer (Fig. 1a). The limiting aperture of the pore is just sufficient to accommodate single-stranded DNA (ssDNA) 6,7 , and the adjacent pore vestibule can accommodate doublestranded (duplex) DNA (dsDNA) 7-9 . In the absence of DNA, the open channel current (I o ) through the α-haemolysin pore is 60 pA at 180 mV applied potential in 0.3 M KCl. DNA capture in the nanopore results in a decrease in the current (I). The DNA resides in the pore for a time (t D ) until it leaves, moving to the trans compartment (Fig. 1b). These two parameters, I and t D , together with current noise, are typically used to report results from nanopore experiments 6,10-19 .We used a nanopore instrument to probe the interaction of the Klenow fragment (KF) of Escherichia coli DNA polymerase I with its DNA substrate. This substrate is a duplex DNA formed by base-pairing of a short ssDNA primer with a longer template DNA. The KF catalyses DNA replication by the sequential addition of nucleotides to the primer strand, dictated by Watson-Crick complementarity to the template strand 20 . In contrast with earlier studies examining Exonuclease I/DNA complexes 4 and EcoRI/DNA complexes 5 , our nanopore Capture and translocation of a model DNA template (14 bp hairpin with a 36-nucleotide 5′ overhang and 2′-3′ dideoxycytidine terminus) resulted in a cluster of events with a median duration of 1 ms and an average blockade amplitude I = 20 pA (Fig. 2a). When the KF (2 μM) was subsequently added to the cis compartment under conditions where catalytic activity had been demonstrated in separate experiments (see Supplementary Information, Fig. S1), a second population of events emerged with a 3-ms median dwell time and a higher blockade current (I = 23 pA, Fig. 2b). This class of events is enzyme-concentration-dependent (see Supplementary Information, Fig. S2 and Table S1), consistent with nanopore capture of a DNA/ KF binary complex.Addition of a deoxynucleotide triphosphat...

Nanopores can be used to analyse DNA by monitoring ion currents as individual strands are captured and driven through the pore in single file order by an applied voltage. Here we show that serial replication of individual DNA templates can be achieved by DNA polymerases held at the α-hemolysin nanopore orifice. Replication is blocked in the bulk phase, and is initiated only after the DNA is captured by the nanopore. We used this method, in concert with active voltage control, to observe DNA replication catalyzed by bacteriophage T7 DNA polymerase (T7DNAP) and by the Klenow fragment of DNA polymerase I (KF). T7DNAP advanced on a DNA template against an 80 mV load applied across the nanopore, and single nucleotide additions were measured on the millisecond time scale for hundreds of individual DNA molecules in series. Replication by KF was not observed when this enzyme was held atop the nanopore orifice at 80 mV applied potential. Sequential nucleotide additions by KF were observed upon controlled voltage reversals.

NA polymerases catalyze templatedependent DNA replication. These enzymes are molecular motors that advance by one nucleotide along template DNA with each catalytic cycle. Kinetic studies of the Klenow fragment of Escherichia coli DNA polymerase I (KF), an A-family polymerase, have shown that in an ordered assembly mechanism KF recognizes the double strandϪsingle strand junction of its DNA substrate to form a binary complex, to which deoxynucleoside triphosphate (dNTP) then binds to form a ternary complex. 1 Crystal structures of A-family polymerases have revealed a conserved catalytic domain that resembles a partially open right hand. 2 The palm subdomain contains residues essential for catalysis, the thumb subdomain positions the primer/template DNA duplex in the active site, and the fingers subdomain is essential for binding dNTP substrates. Structures of binary and ternary complexes show that a conformational transition occurs between these two functional states. 3Ϫ5 In ternary complexes, the fingers subdomain rotates in toward the active site, forming a tight steric fit with the nascent base pair. In the ternary complex, the affinity of KF for its DNA substrate is increased by ϳ5to 8-fold. 6 Nanopore-based measurements have emerged as a single molecule technique for the study of DNA polymerases. 7Ϫ9 Nanoscale pores may be etched in solid-state substrates such as silicon nitride, 10 but the highest precision measurements are made using protein pores inserted in lipid bilayers. 7,11Ϫ14,15 Figure 1a illustrates a single pore formed by an ␣-hemolysin (␣-HL) heptamer. The limiting aperture of the pore is just sufficient to accommodate single-

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