Probing the SELEX Process with Next-Generation Sequencing

Background SELEX is an iterative process in which highly diverse synthetic nucleic acid libraries are selected over many rounds to finally identify aptamers with desired properties. However, little is understood as how binders are enriched during the selection course. Next-generation sequencing offers the opportunity to open the black box and observe a large part of the population dynamics during the selection process. Methodology We have performed a semi-automated SELEX procedure on the model target streptavidin starting with a synthetic DNA oligonucleotide library and compared results obtained by the conventional analysis via cloning and Sanger sequencing with next-generation sequencing. In order to follow the population dynamics during the selection, pools from all selection rounds were barcoded and sequenced in parallel. Conclusions High affinity aptamers can be readily identified simply by copy number enrichment in the first selection rounds. Based on our results, we suggest a new selection scheme that avoids a high number of iterative selection rounds while reducing time, PCR bias, and artifacts

The Retrohoming of Linear Group II Intron RNAs in Drosophila melanogaster Occurs by Both DNA Ligase 4–Dependent and –Independent Mechanisms

Mobile group II introns are bacterial retrotransposons that are thought to have invaded early eukaryotes and evolved into introns and retroelements in higher organisms. In bacteria, group II introns typically retrohome via full reverse splicing of an excised intron lariat RNA into a DNA site, where it is reverse transcribed by the intron-encoded protein. Recently, we showed that linear group II intron RNAs, which can result from hydrolytic splicing or debranching of lariat RNAs, can retrohome in eukaryotes by performing only the first step of reverse splicing, ligating their 3′ end to the downstream DNA exon. Reverse transcription then yields an intron cDNA, whose free end is linked to the upstream DNA exon by an error-prone process that yields junctions similar to those formed by non-homologous end joining (NHEJ). Here, by using Drosophila melanogaster NHEJ mutants, we show that linear intron RNA retrohoming occurs by major Lig4-dependent and minor Lig4-independent mechanisms, which appear to be related to classical and alternate NHEJ, respectively. The DNA repair polymerase θ plays a crucial role in both pathways. Surprisingly, however, mutations in Ku70, which functions in capping chromosome ends during NHEJ, have only moderate, possibly indirect effects, suggesting that both Lig4 and the alternate end-joining ligase act in some retrohoming events independently of Ku. Another potential Lig4-independent mechanism, reverse transcriptase template switching from the intron RNA to the upstream exon DNA, occurs in vitro, but gives junctions differing from the majority in vivo. Our results show that group II introns can utilize cellular NHEJ enzymes for retromobility in higher organisms, possibly exploiting mechanisms that contribute to retrotransposition and mitigate DNA damage by resident retrotransposons. Additionally, our results reveal novel activities of group II intron reverse transcriptases, with implications for retrohoming mechanisms and potential biotechnological applications

Group II Intron-Based Gene Targeting Reactions in Eukaryotes

Mobile group II introns insert site-specifically into DNA target sites by a mechanism termed retrohoming in which the excised intron RNA reverse splices into a DNA strand and is reverse transcribed by the intron-encoded protein. Retrohoming is mediated by a ribonucleoprotein particle that contains the intron-encoded protein and excised intron RNA, with target specificity determined largely by base pairing of the intron RNA to the DNA target sequence. This feature enabled the development of mobile group II introns into bacterial gene targeting vectors ("targetrons") with programmable target specificity. Thus far, however, efficient group II intron-based gene targeting reactions have not been demonstrated in eukaryotes.By using a plasmid-based Xenopus laevis oocyte microinjection assay, we show that group II intron RNPs can integrate efficiently into target DNAs in a eukaryotic nucleus, but the reaction is limited by low Mg(2+) concentrations. By supplying additional Mg(2+), site-specific integration occurs in up to 38% of plasmid target sites. The integration products isolated from X. laevis nuclei are sensitive to restriction enzymes specific for double-stranded DNA, indicating second-strand synthesis via host enzymes. We also show that group II intron RNPs containing either lariat or linear intron RNA can introduce a double-strand break into a plasmid target site, thereby stimulating homologous recombination with a co-transformed DNA fragment at frequencies up to 4.8% of target sites. Chromatinization of the target DNA inhibits both types of targeting reactions, presumably by impeding RNP access. However, by using similar RNP microinjection methods, we show efficient Mg(2+)-dependent group II intron integration into plasmid target sites in zebrafish (Danio rerio) embryos and into plasmid and chromosomal target sites in Drosophila melanogster embryos, indicating that DNA replication can mitigate effects of chromatinization.Our results provide an experimental foundation for the development of group II intron-based gene targeting methods for higher organisms

The development, implementation and evaluation of interventions to reduce workplace sitting: a qualitative systematic review and evidence-based operational framework

Background: Prolonged sitting is associated with increased risks of cardiovascular disease, Type 2 diabetes, some cancers, musculoskeletal disorders and premature mortality. Workplaces contribute to a large proportion of daily sitting time, particularly among office-based workers. Interventions to reduce workplace sitting therefore represent important public health initiatives. Previous systematic reviews suggest such interventions can be effective but have reported wide variations. Further, there is uncertainty as to whether effectiveness in controlled trials can be replicated when implemented outside the research setting. The aims of this review are to identify factors important for the implementation of workplace sitting interventions and to translate these findings into a useful operational framework to support the future implementation of such interventions. Methods: A qualitative systematic review was conducted. Four health and social science databases were searched for studies set in the workplace, with office-based employees and with the primary aim of reducing workplace sitting. Extracted data were primarily from author descriptions of interventions and their implementation. Inductive thematic analysis and synthesis was undertaken. Results: Forty studies met the inclusion criteria. Nine descriptive themes were identified from which emerged three higher-order analytical themes, which related to the development , implementation and evaluation of workplace sitting interventions. Key findings inc luded: the importance of groundi ng interventions in theory; utilising participative approaches during intervention development and implementation; and c onducting comprehensive proce ss and outcome evaluations. There was a general under-reporting of info rmation relating to the context within which workplace sitting interventions were implemented, such as details of local organisation processes and structures, as well as the wider political and economic landscape, which if present would aid the translation of knowledge into “ real-world ” settings. Conclusions: These findings provided the basis for an operational framework, which is a representation of all nine descriptive themes and three higher-order analytical themes, to support workplace sitting intervention development, implementation and evaluation. Once tested and refined, this framework has the potential to be incorporated into a practical toolkit, which could be used by a range of organisations to develop, implement and evaluate their own interventions to reduce workplace sitting time amongst staff

Acceptability and feasibility of a low-cost, theory-based and co-produced intervention to reduce workplace sitting time in desk-based university employees

BACKGROUND: Prolonged sedentary time is linked with poor health, independent of physical activity levels. Workplace sitting significantly contributes to sedentary time, but there is limited research evaluating low-cost interventions targeting reductions in workplace sitting. Current evidence supports the use of multi-modal interventions developed using participative approaches. This study aimed to explore the acceptability and feasibility of a low-cost, co-produced, multi-modal intervention to reduce workplace sitting. METHODS: The intervention was developed with eleven volunteers from a large university department in the UK using participative approaches and “brainstorming” techniques. Main components of the intervention included: emails suggesting ways to “sit less” e.g. walking and standing meetings; free reminder software to install onto computers; social media to increase awareness; workplace champions; management support; and point-of-decision prompts e.g. by lifts encouraging stair use. All staff (n = 317) were invited to take part. Seventeen participated in all aspects of the evaluation, completing pre- and post-intervention sitting logs and questionnaires. The intervention was delivered over four weeks from 7th July to 3rd August 2014. Pre- and post-intervention difference in daily workplace sitting time was presented as a mean ± standard deviation. Questionnaires were used to establish awareness of the intervention and its various elements, and to collect qualitative data regarding intervention acceptability and feasibility. RESULTS: Mean baseline sitting time of 440 min/workday was reported with a mean reduction of 26 ± 54 min/workday post-intervention (n = 17, 95 % CI = −2 to 53). All participants were aware of the intervention as a whole, although there was a range of awareness for individual elements of the intervention. The intervention was generally felt to be both acceptable and feasible. Management support was perceived to be a strength, whilst specific strategies that were encouraged, including walking and standing meetings, received mixed feedback. CONCLUSIONS: This small-scale pilot provides encouragement for the acceptability and feasibility of low-cost, multi-modal interventions to reduce workplace sitting in UK settings. Evaluation of this intervention provides useful information to support participatory approaches during intervention development and the potential for more sustainable low-cost interventions. Findings may be limited in terms of generalisability as this pilot was carried out within a health-related academic setting