A self-replicating molecule directs the covalent assembly of component molecules to form a product that is of identical composition to the parent. When the newly formed product also is able to direct the assembly of product molecules, the self-replicating system can be termed autocatalytic. A self-replicating system was developed based on a ribozyme that catalyzes the assembly of additional copies of itself through an RNA-catalyzed RNA ligation reaction. The R3C ligase ribozyme was redesigned so that it would ligate two substrates to generate an exact copy of itself, which then would behave in a similar manner. This self-replicating system depends on the catalytic nature of the RNA for the generation of copies. A linear dependence was observed between the initial rate of formation of new copies and the starting concentration of ribozyme, consistent with exponential growth. The autocatalytic rate constant was 0.011 min ؊1 , whereas the initial rate of reaction in the absence of pre-existing ribozyme was only 3.3 ؋ 10 ؊11 M⅐min ؊1 . Exponential growth was limited, however, because newly formed ribozyme molecules had greater difficulty forming a productive complex with the two substrates. Further optimization of the system may lead to the sustained exponential growth of ribozymes that undergo self-replication. In living systems, replicative processes transfer genetic information from template nucleic acid molecules to newly synthesized, complementary products. Several nonenzymatic templatedependent ligation systems have been devised to study the role of a template in binding and positioning complementary substrates for covalent bond formation (1-5). These have included simple self-replicating systems of the form A ϩ B 3 T, where A and B are substrates that bind to a complementary template, T, and become joined to form a product molecule that is identical to the template (6-9). The unique aspect of self-replicating systems is that the reaction product has the potential to direct additional reactions. The system is termed autocatalytic when the product is an efficient template, and each covalent bond that is formed generates additional template molecules that can direct further joining reactions. The realization of autocatalytic behavior in a self-replicating system implies that sustained exponential growth may be possible.The self-replicating systems that have been studied to date use template molecules composed of nucleic acids (6, 7, 10), peptides (11-14), or small organic compounds (15-17). The nucleic acid-based systems are the most straightforward and rely on simple Watson-Crick pairing interactions between a short oligonucleotide template and two complementary oligonucleotide substrates (6, 7, 10). The substrates are bound at adjacent positions along the template and are joined through a reaction involving chemical groups at their opposed ends. Peptide-based self-replicating systems are similar, except that the components are oligopeptides that have the capacity to form ␣-helices. The template is the hydrophob...

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The polymerase chain reaction (PCR) is widely used for applications which require a high level of specificity and reliability, such as genetic testing, clinical diagnostics, blood screening, forensics and biodefense. Great improvements to PCR performance have been achieved by the use of Hot Start activation strategies that aim to prevent DNA polymerase extension until more stringent, higher temperatures are reached. Herein we present a novel Hot Start activation approach in PCR where primers contain one or two thermolabile, 4-oxo-1-pentyl (OXP) phosphotriester (PTE) modification groups at 3′-terminal and 3′-penultimate internucleotide linkages. Studies demonstrated that the presence of one or more OXP PTE modifications impaired DNA polymerase primer extension at the lower temperatures that exist prior to PCR amplification. Furthermore, incubation of the OXP-modified primers at elevated temperatures was found to produce the corresponding unmodified phosphodiester (PDE) primer, which was then a suitable DNA polymerase substrate. The OXP-modified primers were tested in conventional PCR with endpoint detection, in one-step reverse transcription (RT)–PCR and in real-time PCR with SYBR Green I dye and Taqman® probe detection. When OXP-modified primers were used as substitutes for unmodified PDE primers in PCR, significant improvement was observed in the specificity and efficiency of nucleic acid target amplification.

For most sample types, the automation of RNA and DNA sample preparation workflows enables high throughput next-generation sequencing (NGS) library preparation. Greater adoption of small RNA (sRNA) sequencing has been hindered by high sample input requirements and inherent ligation side products formed during library preparation. These side products, known as adapter dimer, are very similar in size to the tagged library. Most sRNA library preparation strategies thus employ a gel purification step to isolate tagged library from adapter dimer contaminants. At very low sample inputs, adapter dimer side products dominate the reaction and limit the sensitivity of this technique. Here we address the need for improved specificity of sRNA library preparation workflows with a novel library preparation approach that uses modified adapters to suppress adapter dimer formation. This workflow allows for lower sample inputs and elimination of the gel purification step, which in turn allows for an automatable sRNA library preparation protocol.

In order to study base pairing properties of the amide group in DNA duplexes, a nucleoside analog, 1-(2'-deoxy-beta-D-ribofuranosyl)pyrrole-3-carboxamide, was synthesized by a new route from the ester, methyl 1-(2'-deoxy-3',5'-di-O-p -toluoyl-beta-D-erythro-pentofuranosyl)pyrrole-3-carboxylate, obtained from the coupling reaction between 1-chloro-2-deoxy-3,5-di-O -toluoyl-d-erythropentofuranose and methyl pyrrole-3-carboxylate by treatment with dimethylaluminum amide. 1-(2'-Deoxy-beta-D-ribofuranosyl)pyrrole-3-carboxamide was incorporated into a series of oligodeoxyribonucleotides by solid-phase phosphoramidite technology. The corresponding oligodeoxyribonucleotides with 3-nitropyrrole in the same position in the sequence were synthesized for UV comparison of helix-coil transitions. The thermal melting studies indicate that pyrrole-3-carboxamide, which could conceptually adopt either a dA-like or a dI-like hydrogen bond conformation, pairs with significantly higher affinity to T than to dC. Pyrrole-3-carboxamide further resembles dA in the relative order of its base pairing preferences (T >dG >dA >dC). Theoretical calculations on the model compound N-methylpyrrole-3-carboxamide using density functional theory show little difference in the preference for a syntau versus anti conformation about the bond from pyrrole C3 to the amide carbonyl. The amide groups in both the minimized antitau and syntau conformations are twisted out of the plane of the pyrrole ring by 6-14 degrees. This twist may be one source of destabilization when the amide group is placed in the helix. Another contribution to the difference in stability between the base pairs of pyrrole-3-carboxamide with T and pyrrole-3-carboxamide with C may be the presence of a hydrogen bond in the former involving an acidic proton (N3-H of T).

The development of novel artificial nucleobases and detailed X-ray crystal structures for primer/template/DNA polymerase complexes provide opportunities to assess DNA-protein interactions that dictate specificity. Recent results have shown that base pair shape recognition in the context of DNA polymerase must be considered a significant component. The isosteric azole carboxamide nucleobases (compounds 1-5; ) differ only in the number and placement of nitrogen atoms within a common shape and therefore present unique electronic distributions that are shown to dictate the selectivity of template-directed nucleotide incorporation by DNA polymerases. The results demonstrate how nucleoside triphosphate substrate selection by DNA polymerase is a complex phenomenon involving electrostatic interactions in addition to hydrogen bonding and shape recognition. These azole nucleobase analogs offer unique molecular tools for probing nonbonded interactions dictating substrate selection and fidelity of DNA polymerases.

DNA that encodes elements for degenerate replication events by use of artificial nucleobases offers a versatile approach to manipulating sequences for applications in biotechnology. We have designed a family of artificial nucleobases that are capable of assuming multiple hydrogen bonding orientations through internal bond rotations to provide a means for degenerate molecular recognition. Incorporation of these analogs into a single position of a PCR primer allowed for analysis of their template effects on DNA amplification catalyzed by Thermus aquaticus (Taq) DNA polymerase. All of the nucleobase surrogates have similar shapes but differ by structural alterations that influence their electronic character. These subtle distinctions were able to influence the Taq DNA polymerase dependent incorporation of the four natural deoxyribonucleotides and thus, significantly expand the molecular design possibilities for biochemically functional nucleic acid analogs.

An RNA ligase ribozyme was converted to a corresponding deoxyribozyme through in vitro evolution. The ribozyme was prepared as a DNA molecule of the same sequence, and had no detectable activity. A population of randomized variants of this DNA was constructed and evolved to perform RNA ligation at a rate similar to that of the starting ribozyme. When the deoxyribozyme was prepared as an RNA molecule of the same sequence, it had no detectable activity. Thus, the evolutionary transition from an RNA to a DNA enzyme represents a switch, rather than a broadening, of the chemical basis for catalytic function. This transfer of both information and function is relevant to the transition between two different genetic systems based on nucleic acid-like molecules, as postulated to have occurred during the early history of life on Earth.