Size-expanded ribonucleic acids (xRNAs) : a novel component in siRNAs for investigating the steric restrictions of RISC in RNAi
Abstract/Contents
- Abstract
- This thesis project describes the synthesis and use of size-expanded ribonucleic acids (xRNAs) as unnatural components in small interfering RNAs (siRNAs), which serve as a novel set of steric probes for investigating and identifying size restrictions that exist in the active site of the RNA-induced silencing complex (RISC), a critical protein complex in the RNA interference (RNAi) mechanism. xRNAs are analogous to natural RNAs, in which they retain canonical Watson Crick base-pairing groups, except their nucleobases are expanded linearly by 2.4 Å via benzo-fusion. In this report, the synthesis of the entire set of four xRNA nucleosides and their photophysical properties are described. Two of the xRNA nucleosides (i.e. rxA and rxU) are then converted to their corresponding phosphoramidites for use in automated RNA synthesis to synthesize various sets of xRNA-substituted siRNA strands for biophysical and biological studies. RNAi activity studies in HeLa cells using a firefly/Renilla luciferase dual reporter assay showed that xRNA substitutions in the antisense strand displayed poor RNAi activity in the middle of the sequence (positions 7-14) but were generally well tolerated near the 5'- and 3'-ends. Most significantly, single xRNA substitutions at the 3'-end showed RNAi activity that was more potent than that of unmodified siRNAs. Thermal denaturation studies revealed small changes in melting temperature (+1.4 to --5.0 °C); xRNA substitutions in the middle were found to be more thermodynamically destabilizing whereas the ends were shown to be stabilizing. Serum stability studies showed that xRNA-containing siRNAs have longer half-lives in human serum than unmodified siRNAs. Finally, xRNA-substituted siRNA strands and duplexes showed interesting position-dependent fluorescent properties, while CD spectroscopy experiments revealed that xRNA-substituted siRNAs does not greatly distort the native A-form helical structure of the duplexes. Taken together, the data demonstrate the utility of xRNA nucleobases as size-expanded mechanistic probes for RNAi research.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2012 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Hernandez, Armando Ricardo |
|---|---|
| Associated with | Stanford University, Department of Chemistry |
| Primary advisor | Kool, Eric T |
| Thesis advisor | Kool, Eric T |
| Thesis advisor | Khosla, Chaitan, 1964- |
| Thesis advisor | Wandless, Thomas |
| Advisor | Khosla, Chaitan, 1964- |
| Advisor | Wandless, Thomas |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Armando Ricardo Hernández. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2012. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2012 by Armando Ricardo Hernandez
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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