Lipid-assisted synthesis of RNA-like polymers from mononucleotides.

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.

Single-molecule analysis of DNA-protein complexes using nanopores.

We present a method for rapid measurement of DNA-protein interactions using voltage-driven threading of single DNA molecules through a protein nanopore. Electrical force applied to individual ssDNA-exonuclease I complexes pulls the two molecules apart, while ion current probes the dissociation rate of the complex. Nanopore force spectroscopy (NFS) reveals energy barriers affecting complex dissociation. This method can be applied to other nucleic acid-protein complexes, using protein or solid-state nanopore devices.

Cheminformatics methods for novel nanopore analysis of HIV DNA termini.

BACKGROUND: Channel current feature extraction methods, using Hidden Markov Models (HMMs) have been designed for tracking individual-molecule conformational changes. This information is derived from observation of changes in ionic channel current blockade "signal" upon that molecule's interaction with (and occlusion of) a single nanometer-scale channel in a "nanopore detector". In effect, a nanopore detector transduces single molecule events into channel current blockades. HMM analysis tools described are used to help systematically explore DNA dinucleotide flexibility, with particular focus on HIV's highly conserved (and highly flexible/reactive) viral DNA termini. One of the most critical stages in HIV's attack is the binding between viral DNA and the retroviral integrase, which is influenced by the dynamic-coupling induced high flexibility of a CA/TG dinucleotide positioned precisely two base-pairs from the blunt terminus of the duplex viral DNA. This suggests the study of a family of such CA/TG dinucleotide molecules via nanopore measurement and cheminformatics analysis. RESULTS: HMMs are used for level identification on the current blockades, HMM/EM with boosted variance emissions are used for level projection pre-processing, and time-domain FSAs are used to parse the level-projected waveform for kinetic information. The observed state kinetics of the DNA hairpins containing the CA/TG dinucleotide provides clear evidence for HIV's selection of a peculiarly flexible/interactive DNA terminus.