BHQ1 aptamer, and the construction of RT aptamers that hybrid-
ize to the target RNA. This imaging technology could provide new
insights into RNA function and gene expression.2 Materials
All solutions are prepared using ultrapure water (purchased from
Merck Millipore) and analytical grade reagents. Reagents are
prepared and stored at room temperature unless otherwise indi-
cated. Distilled water and special grade solvents and reagents are
used for chemical syntheses.2.1 Reagents 1. EAH Sepharose™4B resin (General Electric).
- BHQ1 carboxylic acid (e.g., Biosearch Technologies) (see
Note 1). - BHQ1 amine (e.g., Biosearch Technologies) (seeNote 1).
- 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride n-hydrate (DMT-MM; e.g., Wako Pure Chemical
Industries). - Triethylamine.
- Acetic acid.
- Dioxane.
- Methanol.
- Fmoc-β-Ala-Wang Resin (0.18 meq/g; e.g., Peptides
International).
10.N,N-dimethylformamide (DMF). - Dichloromethane.
- 20 v/v % piperidine in DMF (e.g., Watanabe Chemical
Industries). - Fmoc-amido-dPEG4™-acid (e.g., Quanta BioDesign).
14.N,N^0 -diisopropylcarbodiimide (e.g., Wako Pure Chemical
Industries). - 1-Hydroxybenzotriazole monohydrate (HOBt; e.g., Wako
Pure Chemical Industries). - Cy3-NHS-ester (e.g., Lumiprobe) (seeNote 1).
- Acetic anhydride (e.g., Tokyo Chemical Industry) (seeNote 2).
- Trifluoroacetic acid (TFA; e.g., Wako Pure Chemical
Industries). - Triisopropylsilane (e.g., Tokyo Chemical Industry).
20.O-(6-chlorobenzotriazol-1-yl)-N,N,N^0 ,N^0 -tetramethyluro-
nium hexafluorophosphate (HCTU; e.g., Wako Pure Chemical
Industries).
306 Shin-ichi Sato et al.