The preparation of site-specifically modified riboswitch domains as an example for enzymatic ligation of chemically synthesized RNA fragments.
This protocol describes an efficient method for the preparation of riboswitch domains comprising up to approximately 200 nt containing site-specific nucleoside modifications. The strategy is based on enzymatic ligation of chemically synthesized RNA fragments. The design of ligation sites strictly follows the criterion that all fragments comprise less than approximately 50 nt. This allows the researcher to rely on custom synthesis services and to utilize the large pool of commercially available, functionalized nucleoside phosphoramidites for solid-phase RNA synthesis. Importantly, this design renders utmost flexibility to position a chemical modification (e.g., a fluorescence label) within the RNA. Selection of the appropriate ligation type (using T4 RNA or T4 DNA ligase) is subordinate to the criteria above and is detailed in the protocol. The whole concept is demonstrated for 2-aminopurine containing thiamine pyrophosphate responsive riboswitch domains that are applied in fluorescence spectroscopic folding studies. Labeled samples in 5-35 nmol quantities are obtained within 3-4 d, not including the time for fragment synthesis.
Lang
Kathrin
K
Institute of Organic Chemistry, Center for Molecular Biosciences Innsbruck, Leopold Franzens University, Innrain 52a, A-6020 Innsbruck, Austria.
Micura
Ronald
R
D015075:2-Aminopurine / Q000737:chemistry / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary / Q000235:genetics*; D012313:RNA / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013835:Thiamine Pyrophosphate / Q000378:metabolism
D015075:2-Aminopurine; D012313:RNA; D013835:Thiamine Pyrophosphate