DESIGN OF NEW NUCLEO · Design of new nucleosides for DNA interactions and fluorescent studies
FP6 — Marie Curie Actions (Human Resources and Mobility)
- Duration
- 2005-01-01 → 2007-12-31
- EU contribution
- €244,369
- Participants
- 2
- Scheme
- OIF
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Results in brief
Final Activity Report Summary - DESIGN OF NEW NUCLEO (Design of new nucleosides for DNA interactions and fluorescent studies)
This project is focus on the study of the effect of chemically modified RNAs in RNA interference. Specifically, the project wanted to address the role of the hydrogen bonding stabilisation and steric effects on RNA interference. We prepared several non-polar isosteres of natural nucleosides, these molecules are very close in size and shape than their natural counterparts but lack the polar groups involved in base pairing. These molecules are good probes to study the importance of hydrogen bonding interaction in biological systems. In this case we wanted to study this interaction in a gene regulation mechanism known as RNA interference. This process can knock down the expression of a specific protein by targeting the corresponding mRNA sequence. This process is triggered when a doubled stranded RNA (siRNA) reach the cytoplasm, then one of the strands known as guide is incorporated into a protein complex (RISC), which then cleaves the complementary sequence present in the mRNA. In this project, we have incorporated several analogs to different positions of two different RNA sequences. After purification of the modified RNA strand they were annealed with the complementary sequence and the corresponding thermal stability was determined. The RNA interference experiments were carried out on HeLa cells using the dual-luciferase system. The results have shown that the hydrogen bonding stabilisation is not required at every position; moreover at position 7 of the guide strand the activity is high despite the low thermal stability. Therefore we have carried out selectivity studies at position 7 utilising mutated target sequences. The results of this set of experiments have shown that steric effects are critical in selectivity. This work has been published in Angewandte Chemie Int. Ed. 2006, 45, 4994-4997.
Data: CORDIS, © European Union
Project objective
The strategy of replacing DMA natural bases by non-polar surrogates has opened ways to add new functions to the DNA structure, to probe protein-DNA interactions, to test DMA repair enzymes, to stabilize structure, and to probe the internal features of the helix. Also, different fluorescent species named fluorosides, which have been shown an increasingly useful tool for bio-molecular analysis and is applied widely in the biosciences.This project wants to develop a new series of nucleosides with different characteristics. One group will have different steric demanding nucleosides which will be used to study the interaction with DNA polymerase enzyme and the other group will have fluorescent activity which will be evaluated as a molecular tags.
Original text from CORDIS.
Participants
- UNIVERSIDAD AUTONOMA DE MADRID · MADRIDCoordinatorSpain
- DEPARTMENT OF CHEMISTRY STANFORD UNIVERSITY · STANFORDCity levelUnited States
Links
Data: CORDIS, © European Union