Diazo-based precursors of photolabile groups have been used extensively for modifying nucleic acids, with the intention of toggling biological processes with light. These processes include transcription, translation and RNA interference. In these cases, the photolabile groups have been typically depicted as modifying the phosphate backbone of RNA and DNA. In this work we find that these diazo-based reagents in fact react very poorly with backbone phosphates. Instead, they show a remarkable specificity for terminal phosphates and very modest modification of the nucleobases. Furthermore, the photo deprotection of these terminal modifications is shown to be much more facile than nucleobase modified sites. In this study we have characterized this regiospecificity using RNA duplexes and model nucleotides, analyzed using LC/MS/MS. We have also applied this understanding of the regio-specificity to our technique of light activated RNA interference (LARI). We examined 27-mer double-stranded precursors of siRNA (‘dsRNA’), and have modified them using the photo-cleavable di-methoxy nitro phenyl ethyl group (DMNPE) group. By incorporating terminal phosphates in the dsRNA, we are able to guide DMNPE to react at these terminal locations. These modified dsRNA duplexes show superior performance to our previously described DMNPE-modified siRNA, with the range of expression that can be toggled by light increasing by a factor of two.
An LC-MS/MS method to measure ribociclib in mouse plasma and Ringer’s solution was successfully developed and validated. Reverse phase chromatography was performed with gradient elution using C18 (100A, 50x4.6 mm, 3μ) and C8-A (50x 2.0 mm, 5 μ) columns for plasma and Ringer’s samples, respectively. Mouse plasma samples were extracted using solid phase extraction method, whereas no extraction was required for the Ringer’s solution samples. Analytes were detected using positive ion MRM mode. The precursor to product ions (Q1→Q3) selected for ribociclib and d6-ribociclib were (m/z) 435.2 → 252.1 and 441.2 → 252.1, respectively. The linear range of quantification of ribociclib was 62.5–10000 ng/ml for plasma method and 0.1–100 ng/ml for Ringer’s solution method. The results for the inter-day and intra-day accuracy and precision of quality control samples were within the acceptable range. The lower limit of quantitation (LLOQ) for plasma and Ringer’s samples were 62.5 ng/ml (S/N > 30) and 0.1 ng/ml (S/N > 13), respectively, whereas the limit of detection (LOD) was 6.9 ng/ml (S/N > 7) and 0.05 ng/ml (S/N > 3), respectively. The developed methods were successfully applied to the analysis of ribociclib in mouse plasma and dialysate samples collected during a cerebral microdialysis study of ribociclib in a non-tumor bearing mouse.
Incorporating phosphorothioate groups into dsRNA both stabilizes them towards degradation by serum enzymes, as well as improves them as the basis for light-activated RNA interference.
Light-activated RNA interference (LARI) is an effective way to control gene expression with light. This, in turn, allows for the spacing, timing and degree of gene expression to be controlled by the spacing, timing and amount of light irradiation. The key mediators of this process are siRNA or dsRNA that have been modified with four photocleavable groups of dimethoxy nitro phenyl ethyl (DMNPE), located on the four terminal phosphate groups of the duplex RNA. These mediators can be easily synthesized and purified using two readily available products: synthetic RNA oligonucleotides and DMNPE-hydrazone. The synthesis of the tetra-DMNPE-modified duplex RNA is made possible by a remarkable regiospecificity of DMNPE for terminal phosphates (over internal phosphates or nucleobases) that we have previously identified. The four installed DMNPE groups effectively limit RNAi until irradiation cleaves them, releasing native, active siRNA. By using the described protocol, any process that is mediated by RNAi can be controlled with light. Although other methods exist to control gene expression with light by using specialized reagents, this method requires only two commercially available products. The protocol takes ∼3 d in total for the preparation of modified RNA.
For the first time, cells have been patterned on surfaces through the spatial manipulation of native gene expression. By manipulating the inherent biology of the cell, as opposed to the chemical nature of the surfaces they are attached to, we have created a potentially more flexible way of creating patterns of cells that does not depend on the substrate. This was accomplished by bringing an siRNA that targets the expression of pten under the control of light, by modifying it with photocleavable groups. This pten-targeting siRNA has been previously demonstrated to induce dissociation of cells from surfaces. We modified this siRNA with dimethoxy nitro phenyl ethyl photocleavable groups (DMNPE) to allow the activity of the siRNA, and hence pten knockdown, to be toggled with light. Using this approach we demonstrated light dependent cell dissociation only with a DMNPE modified siRNA that targets pten and not with control siRNAs. In addition we demonstrated the ability to make simple patterns of cells through the application of masks during irradiation.
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