Improved deoxyribozymes for synthesis of covalently branched DNA and RNA

A covalently branched nucleic acid can be synthesized by joining the 2′-hydroxyl of the branch-site ribonucleotide of a DNA or RNA strand to the activated 5′-phosphorus of a separate DNA or RNA strand. We have previously used deoxyribozymes to synthesize several types of branched nucleic acids for experiments in biotechnology and biochemistry. Here, we report in vitro selection experiments to identify improved deoxyribozymes for synthesis of branched DNA and RNA. Each of the new deoxyribozymes requires Mn2+ as a cofactor, rather than Mg2+ as used by our previous branch-forming deoxyribozymes, and each has an initially random region of 40 rather than 22 or fewer combined nucleotides. The deoxyribozymes all function by forming a three-helix-junction (3HJ) complex with their two oligonucleotide substrates. For synthesis of branched DNA, the best new deoxyribozyme, 8LV13, has kobs on the order of 0.1 min−1, which is about two orders of magnitude faster than our previously identified 15HA9 deoxyribozyme. 8LV13 also functions at closer-to-neutral pH than does 15HA9 (pH 7.5 versus 9.0) and has useful tolerance for many DNA substrate sequences. For synthesis of branched RNA, two new deoxyribozymes, 8LX1 and 8LX6, were identified with broad sequence tolerances and substantial activity at pH 7.5, versus pH 9.0 for many of our previous deoxyribozymes that form branched RNA. These experiments provide new, and in key aspects improved, practical catalysts for preparation of synthetic branched DNA and RNA

Structure of the ATP synthase catalytic complex (F(1)) from Escherichia coli in an autoinhibited conformation.

ATP synthase is a membrane-bound rotary motor enzyme that is critical for cellular energy metabolism in all kingdoms of life. Despite conservation of its basic structure and function, autoinhibition by one of its rotary stalk subunits occurs in bacteria and chloroplasts but not in mitochondria. The crystal structure of the ATP synthase catalytic complex (F(1)) from Escherichia coli described here reveals the structural basis for this inhibition. The C-terminal domain of subunit ɛ adopts a heretofore unknown, highly extended conformation that inserts deeply into the central cavity of the enzyme and engages both rotor and stator subunits in extensive contacts that are incompatible with functional rotation. As a result, the three catalytic subunits are stabilized in a set of conformations and rotational positions distinct from previous F(1) structures

Padlock Probes and Rolling Circle Amplification : New Possibilities for Sensitive Gene Detection

A series of novel methods for detection of known sequence variants in DNA, in particular single nucleotide polymorphism, using padlock probes and rolling circle replication are presented. DNA probes that can be circularized – padlock probes – are ideal for rolling circle replication. Circularized, but not unreacted probes, can generate powerful signal amplification by allowing the reacted probes to template a rolling circle replication (RCR) reaction. However, when hybridized and ligated to a target DNA molecule with no nearby ends, the probes are bound to the target sequence, inhibiting the RCR reaction is. This problem can be solved by generating a branched DNA probe with two 3’ arms such that the probes may be circularized while leaving the second 3’ arm as a primer for the RCR reaction. We describe how T4 DNA ligase can be used for efficient construction of DNA molecules having one 5’ end but two distinct 3’ ends that extend from the 2’ and 3’ carbons of an internal nucleotide. An even stronger approach to circumvent the topological problem that can inhibit RCR is to restriction digest the template downstream of the padlock recognition site. By using Phi 29 DNA polymerase with efficient 3’ exonuclease and strand displacement activity, the template strand can then be used to prime the RCR reaction. The amplified molecule is contiguous with the target DNA, generating an anchored localized signal. The kinetics of the reaction was investigated by following the reaction in real-time using molecular beacon probes. Localized RCR signal were obtained on DNA arrays, allowing detection of as little as 104-105 spotted molecules, of either single- or double-stranded M13 DNA, in a model experiment. We have also established a serial rolling circle amplification procedure. By converting rolling circle products to a second and even third generation of padlock probes the signal was amplified thousand-fold per generation. This procedure provides sufficient sensitivity for detection of single-copy gene sequences in 50 ng of human genomic DNA, and large numbers of probes were amplified in parallel with excellent quantitative resolution

Skogsformande - om bilden av skogen som rum eller resurs, och en jakt på en tredje dröm

This work investigates, from an architect’s perspective, images of the forest as a space and as a resource. In a personal exploration, the author’s own relationship to forests, as well as cultural and scholarly conceptions of what the forest is and means are examined and questioned, connected and contrasted. Seemingly uncomplicated popular images that are shaping our view of the forest as well as the living forests themselves, turn out to be both complex and conflicting. The idea of wood as an eco-friendly building material, and marketing slogans such as the “green factory”, are set up against the realities of industrial forestry and forest devastation. The sentimental human desire to regain or return to the forest is contrasted with our species’ history of dominating it. And the romantic idea of the forest as a static epitome of Nature is challenged by the shifting shapes of forests and the inescapability of human interference. Recurring throughout this inquiry is the problematic dual role of the forest as both space and resource; nature and culture. In an attempt to find a forest path between and beyond divisive dualities, the author is escorted on her journey by the philosophy of Donna Haraway. Haraway’s calls to “stay with the trouble” and “becoming-with” may inspire new alliances between humans, trees and the other life forms of the woods. Should we manage to discover the forests in ourselves, and vice-versa, we might also become able to create images and spaces that - instead of separation, exploitation and uniformity - are shaped by and shape connectedness, co-dependency and complexity. The written report of this work is supplemented by recorded talks with ten different professionals who in one way or another have strong relationships to the forest. The talks, which are available online, are cited throughout the written text and comprise an equal part of this work as the report itself

What Drives Liquefied Natural Gas Imports in Europe?

This paper studied the extensive margin (EM) and intensive margin (IM)of liquefied natural gas(LNG) imports in Europe over the period 1996-2015. Two econometric models were used, a prob it estimation for the EM and an OLS for the IM. A time-varying approach was conducted to analyse the stability of the models in the studied time frame. The models were constructed through the application of known determinants of LNG trade as well as new factors that previously was unused in the investigation of LNG trade. The results indicated an overall stable EM, but a highly varying IM over the period. The findings inform that the EM is driven by income, diversification and lower bounds technological development and we found that itis inhibited by pipeline imports, domestic production and higher bounds technological development. The IM is determined by favourable pricing opportunities, lower bounds technological development and the diversification aspect of LNG. IM is negatively affected by domestic natural gas production and the higher bounds of technological development

What Drives Liquefied Natural Gas Imports in Europe?

This paper studied the extensive margin (EM) and intensive margin (IM)of liquefied natural gas(LNG) imports in Europe over the period 1996-2015. Two econometric models were used, a prob it estimation for the EM and an OLS for the IM. A time-varying approach was conducted to analyse the stability of the models in the studied time frame. The models were constructed through the application of known determinants of LNG trade as well as new factors that previously was unused in the investigation of LNG trade. The results indicated an overall stable EM, but a highly varying IM over the period. The findings inform that the EM is driven by income, diversification and lower bounds technological development and we found that itis inhibited by pipeline imports, domestic production and higher bounds technological development. The IM is determined by favourable pricing opportunities, lower bounds technological development and the diversification aspect of LNG. IM is negatively affected by domestic natural gas production and the higher bounds of technological development

What Drives Liquefied Natural Gas Imports in Europe?

This paper studied the extensive margin (EM) and intensive margin (IM)of liquefied natural gas(LNG) imports in Europe over the period 1996-2015. Two econometric models were used, a prob it estimation for the EM and an OLS for the IM. A time-varying approach was conducted to analyse the stability of the models in the studied time frame. The models were constructed through the application of known determinants of LNG trade as well as new factors that previously was unused in the investigation of LNG trade. The results indicated an overall stable EM, but a highly varying IM over the period. The findings inform that the EM is driven by income, diversification and lower bounds technological development and we found that itis inhibited by pipeline imports, domestic production and higher bounds technological development. The IM is determined by favourable pricing opportunities, lower bounds technological development and the diversification aspect of LNG. IM is negatively affected by domestic natural gas production and the higher bounds of technological development