Overcoming nucleotide bias in the nonenzymatic copying of RNA templates.

The RNA World hypothesis posits that RNA was the molecule of both heredity and function during the emergence of life. This hypothesis implies that RNA templates can be copied, and ultimately replicated, without the catalytic aid of evolved enzymes. A major problem with nonenzymatic template-directed polymerization has been the very poor copying of sequences containing rA and rU.

Solid-State Single-Molecule Sensing with the Electronic Life-Detection Instrument for Enceladus/Europa (ELIE).

Growing evidence of the potential habitability of Ocean Worlds across our solar system is motivating the advancement of technologies capable of detecting life as we know it-sharing a common ancestry or physicochemical origin with life on Earth-or don't know it, representing a distinct emergence of life different than our one known example. Here, we propose the Electronic Life-detection Instrument for Enceladus/Europa (ELIE), a solid-state single-molecule instrument payload that aims to search for life based on the detection of amino acids and informational polymers (IPs) at the parts per billion to trillion level. As a first proof-of-principle in a laboratory environment, we demonstrate the single-molecule detection of the amino acid L-proline at a 10 μM concentration in a compact system.

Freeze-thaw cycles enable a prebiotically plausible and continuous pathway from nucleotide activation to nonenzymatic RNA copying.

Nonenzymatic template-directed RNA copying using chemically activated nucleotides is thought to have played a key role in the emergence of genetic information on the early Earth. A longstanding question concerns the number and nature of different environments that might have been necessary to enable all of the steps from nucleotide synthesis to RNA copying. Here we explore three sequential steps from this overall pathway: nucleotide activation, synthesis of imidazolium-bridged dinucleotides, and template-directed RNA copying.

Competition between bridged dinucleotides and activated mononucleotides determines the error frequency of nonenzymatic RNA primer extension.

Nonenzymatic copying of RNA templates with activated nucleotides is a useful model for studying the emergence of heredity at the origin of life. Previous experiments with defined-sequence templates have pointed to the poor fidelity of primer extension as a major problem. Here we examine the origin of mismatches during primer extension on random templates in the simultaneous presence of all four 2-aminoimidazole-activated nucleotides.

Potentially Prebiotic Activation Chemistry Compatible with Nonenzymatic RNA Copying.

The nonenzymatic replication of ribonucleic acid (RNA) may have enabled the propagation of genetic information during the origin of life. RNA copying can be initiated in the laboratory with chemically activated nucleotides, but continued copying requires a source of chemical energy for nucleotide activation. Recent work has illuminated a potentially prebiotic cyanosulfidic chemistry that activates nucleotides, but its application to nonenzymatic RNA copying had not been demonstrated.

Deep sequencing of non-enzymatic RNA primer extension.

Life emerging in an RNA world is expected to propagate RNA as hereditary information, requiring some form of primitive replication without enzymes. Non-enzymatic template-directed RNA primer extension is a model of the copying step in this posited form of replication. The sequence space accessed by primer extension dictates potential pathways to self-replication and, eventually, ribozymes.

In vitro selection of ribozyme ligases that use prebiotically plausible 2-aminoimidazole-activated substrates.

The hypothesized central role of RNA in the origin of life suggests that RNA propagation predated the advent of complex protein enzymes. A critical step of RNA replication is the template-directed synthesis of a complementary strand. Two experimental approaches have been extensively explored in the pursuit of demonstrating protein-free RNA synthesis: template-directed nonenzymatic RNA polymerization using intrinsically reactive monomers and ribozyme-catalyzed polymerization using more stable substrates such as biological 5'-triphosphates.

The dynamics of eukaryotic replication initiation: origin specificity, licensing, and firing at the single-molecule level.

Eukaryotic replication initiation is highly regulated and dynamic. It begins with the origin recognition complex (ORC) binding DNA sites called origins of replication. ORC, together with Cdc6 and Cdt1, mediate pre-replicative complex (pre-RC) assembly by loading a double hexamer of Mcm2-7: the core of the replicative helicase.

DNA curtains: novel tools for imaging protein-nucleic acid interactions at the single-molecule level.

Interactions between proteins and nucleic acids are at the molecular foundations of most key biological processes, including DNA replication, genome maintenance, the regulation of gene expression, and chromosome segregation. A complete understanding of these types of biological processes requires tackling questions with a range of different techniques, such as genetics, cell biology, molecular biology, biochemistry, and structural biology. Here, we describe a novel experimental approach called "DNA curtains" that can be used to complement and extend these more traditional techniques by providing real-time information about protein-nucleic acid interactions at the level of single molecules.

Towards physiological complexity with in vitro single-molecule biophysics.

Single-molecule biology has matured in recent years, driven to greater sophistication by the development of increasingly advanced experimental techniques. A progressive appreciation for its unique strengths is attracting research that spans an exceptionally broad swath of physiological phenomena--from the function of nucleosomes to protein diffusion in the cell membrane. Newfound enthusiasm notwithstanding, the single-molecule approach is limited to an intrinsically defined set of biological questions; such limitation applies to all experimental approaches, and an explicit statement of the boundaries delineating each set offers a guide to most fruitfully orienting in vitro single-molecule research in the future.

Unusual structures are present in DNA fragments containing super-long Huntingtin CAG repeats.

Background: In the R6/2 mouse model of Huntington's disease (HD), expansion of the CAG trinucleotide repeat length beyond about 300 repeats induces a novel phenotype associated with a reduction in transcription of the transgene.

Methodology/principal Findings: We analysed the structure of polymerase chain reaction (PCR)-generated DNA containing up to 585 CAG repeats using atomic force microscopy (AFM). As the number of CAG repeats increased, an increasing proportion of the DNA molecules exhibited unusual structural features, including convolutions and multiple protrusions.

The importance of surfaces in single-molecule bioscience.

The last ten years have witnessed an explosion of new techniques that can be used to probe the dynamic behavior of individual biological molecules, leading to discoveries that would not have been possible with more traditional biochemical methods. A common feature among these single-molecule approaches is the need for the biological molecules to be anchored to a solid support surface. This must be done under conditions that minimize nonspecific adsorption without compromising the biological integrity of the sample.