Synthetic construct
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA116763 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Cervical cancer is a global public health subject as it affects women in the reproductive ages, and accounts for the second largest burden among cancer patients worldwide with an unforgiving 50% mortality rate. Poor awareness and access to effective diagnosis have led to this enormous disease burden, calling for point-of-care, minimally invasive diagnosis methods. Here, an end-to-end quantitative approach for a new kind of diagnosis has been developed, comprising identification of optimal biomarkers, design of the sensor, and simulation of the diagnostic circuit. Using miRNA expression data in the public domain, we identified circulating miRNA biomarkers specific to cervical cancer using multi-tier screening. Synthetic riboregulators called toehold switches specific for the biomarker panel were then designed. To predict the dynamic range of toehold switches for use in genetic circuits as biosensors, we developed a generic grammar of these switches, and built a multivariate linear regression model using thermodynamic features derived from RNA secondary structure and interaction. The model yielded predictions of toehold efficacy with an adjusted R2 = 0.59. Reaction kinetics modelling was performed to predict the sensitivity of the second-generation toehold switches to the miRNA biomarkers. Simulations showed a linear response between 10nM and 100nM before saturation. Our study demonstrates an end-to-end workflow for the efficient design of genetic circuits geared towards the effective detection of unique genomic signatures that would be increasingly important in today’s world. The approach has the potential to direct experimental efforts and minimise costs. All resources are provided open-source (https://github.com/igem2019) under GNU GPLv3 licence.
2022-05-05 | MODEL2107150001 | BioModels
Project description:Dynamic multilayer DNA based data storage
| PRJNA292092 | ENA
Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. The linear dynamic range and sensitivity of the microarray was measured by hybridizing dilution series of a Universal Reference (UR), an equimolar mixture of synthetic miRNAs. The UR was hybridized with 10,000 to 1 amol of each individual miRNA. Each individual miRNA and 1 fmol of each of 18 RNA oligonucleotides reverse complement to miRControl 3 probes was fluorescently labelled by 3â ligation. The RNA mix was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool.
2010-06-20 | E-GEOD-15834 | biostudies-arrayexpress
Project description:Post-transcriptional generation of miRNA variants by multiple nucleotidyl transferases contributes to miRNA transcriptome complexity (Validation 2)
| PRJNA142159 | ENA
Project description:Post-transcriptional generation of miRNA variants by multiple nucleotidyl transferases contributes to miRNA transcriptome complexity (Validation 1)
| PRJNA142157 | ENA
Project description:The short length of miRNAs results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here we developed a new method based not only to the hybridization process that presents the limits before described, but integrating the hybridization to an enzymatic reaction. Moreover we introduced spike-in in the hybridization-enzymatic reaction allowing the quantification of miRNAs respect to them, canceling biases related to sequence, labeling, or hybridization. An alternative method for the absolute miRNA quantization was recently proposed by Bissels (Absolute quantification of microRNAs by using a universal reference. RNA). It was based on the Absolute quantification of microRNAs by using a universal reference consisting of 954 synthetic human, mouse, rat, and viral miRNAs, with each individual oligoribonucleotide present in equimolar concentrations with tested miRNAs. Thereby, any single miRNA detected on a microarray can be quantified by directly comparing its signal intensity with the one obtained by the same miRNA sequence present in the universal reference adjusting for biases related to sequence, labeling, hybridization, or signal detection. Our method allowed the detection of a comparable concentration of miRNA (10-18 moles to 10-14 moles in a linear range) (see Figure), but allows controlling the hybridization quality and reproducibility basing on the results of the interpolation of the spike-in dependent curve. Moreover, our method does not influenced by phenomena imputable to different labeling process due to different sequences because labeling was due only to the incorporation of biotin-d(A) if the hybridized miRNA acted as primer for the klenow enzyme. This method allowed the discussion of miRNA genes expression in 14 different tissues relating it with tissue anatomical proximity and functional similarity.
2014-03-31 | GSE28140 | GEO
Project description:The short length of miRNAs results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here we developed a new method based not only to the hybridization process that presents the limits before described, but integrating the hybridization to an enzymatic reaction. Moreover we introduced spike-in in the hybridization-enzymatic reaction allowing the quantification of miRNAs respect to them, canceling biases related to sequence, labeling, or hybridization. An alternative method for the absolute miRNA quantization was recently proposed by Bissels (Absolute quantification of microRNAs by using a universal reference. RNA). It was based on the Absolute quantification of microRNAs by using a universal reference consisting of 954 synthetic human, mouse, rat, and viral miRNAs, with each individual oligoribonucleotide present in equimolar concentrations with tested miRNAs. Thereby, any single miRNA detected on a microarray can be quantified by directly comparing its signal intensity with the one obtained by the same miRNA sequence present in the universal reference adjusting for biases related to sequence, labeling, hybridization, or signal detection. Our method allowed the detection of a comparable concentration of miRNA (10^-18 moles to 10^-14 moles in a linear range), but allows controlling the hybridization quality and reproducibility basing on the results of the interpolation of the spike-in dependent curve. Moreover, our method does not influenced by phenomena imputable to different labeling process due to different sequences because labeling was due only to the incorporation of biotin-d(A) if the hybridized miRNA acted as primer for the klenow enzyme. This method allowed the discussion of miRNA genes expression in 9 different tissues relating them with tissue functionality in the cardiocirculatory system.
2014-04-30 | E-MTAB-1938 | biostudies-arrayexpress
Project description:Substrate selectivity for semisynthetic CK2 proteins with various posttranslational modifications
| PRJNA156597 | ENA
Project description:An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments
| PRJNA268435 | ENA