Dataset Information

The effects of stem length and core placement on shRNA activity.

ABSTRACT:

Background

Expressed short hairpin RNAs (shRNA) used in mammalian RNA interference (RNAi) are often designed around a specific short interfering RNA (siRNA) core. Whilst there are algorithms to aid siRNA design, hairpin-specific characteristics such as stem-length and siRNA core placement within the stem are not well defined.

Results

Using more than 91 hairpins designed against HIV-1 Tat and Vpu, we investigated the influence of both of these factors on suppressive activity, and found that stem length does not correspond with predictable changes in suppressive activity. We also detected multiple processed products for all stem lengths tested. However, the entire length of the hairpin stem was not equally processed into active products. As such, the placement of the siRNA core at the base terminus was critical for activity.

Conclusion

We conclude that there is no fixed correlation between stem length and suppressive activity. Instead, core selection and placement likely have a greater influence on the effectiveness of shRNA-based silencing.

SUBMITTER: McIntyre GJ

PROVIDER: S-EPMC3175162 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Publications

The effects of stem length and core placement on shRNA activity.

McIntyre Glen J GJ Yu Yi-Hsin YH Lomas Mehnaaz M Fanning Gregory C GC

BMC molecular biology 20110808

<h4>Background</h4>Expressed short hairpin RNAs (shRNA) used in mammalian RNA interference (RNAi) are often designed around a specific short interfering RNA (siRNA) core. Whilst there are algorithms to aid siRNA design, hairpin-specific characteristics such as stem-length and siRNA core placement within the stem are not well defined.<h4>Results</h4>Using more than 91 hairpins designed against HIV-1 Tat and Vpu, we investigated the influence of both of these factors on suppressive activity, and f ...[more]

PMID: 21819628

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Project description:Vaccination with Bacillus Calmette-Guérin (BCG) induces long-term innate immune memory, also called trained immunity, characterized by metabolic and epigenetic changes leading to enhanced responsiveness upon exposure to heterologous pathogens. BCG vaccination was also shown to reduce systemic inflammation. Thus, BCG counteracts two significant immunological changes associated with aging: impaired responsiveness and elevated systemic inflammation. However, if and how BCG impacts other aging-related processes in immune cells, such as telomere shortening, remains unexplored. In this study, we investigated the transcriptional impact of BCG training on telomere maintenance-related genes through RNA sequencing and determined average telomere length from whole blood via RT-qPCR before and three months after BCG vaccination in two independent human cohorts. Trained immunity response was measured by ex-vivo cytokine production induced by a heterologous stimulus three months after vaccination compared to baseline. In addition, we examined the effects of BCG on telomerase activation using an in vitro trained immunity model. In vitro BCG training upregulated processes related to telomere maintenance and telomerase localization. In vivo, we observed shorter telomeres three months after BCG vaccination in both studies. Interestingly, the induction of a trained immunity response by BCG was correlated to the change in telomere length: more telomere shortening was observed in trained immunity non-responder individuals, particularly in males. Higher testosterone concentrations before vaccination were linked to more telomere loss. In vitro, BCG training of human monocytes activated the telomerase enzyme, predominantly in females, an effect that was blocked by exogenous testosterone treatment. Overall, this study reports sex-specific long-term impacts of BCG vaccination on telomerase activity and telomere maintenance. These data add to the arguments that BCG vaccination impacts aging mechanisms, which warrants more investigation.

Dataset Information

The effects of stem length and core placement on shRNA activity.

Background

Results

Conclusion

Publications

The effects of stem length and core placement on shRNA activity.

Similar Datasets

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