21.N,N-diisopropylethylamine (e.g., Wako Pure Chemical
Industries).- PCR Master mix (e.g., Toyobo KOD FX Neo).
- 3 M sodium acetate buffer Solution pH 5.2.
- 70% ethanol.
- TE Buffer Solution pH 8.0.
- T7 in vitro transcription kit including DNase (Thermo Fisher
Scientific MEGAscript®T7 Kit). - 10 M ammonium acetate solution pH 7.0.
- DNA purification columns (e.g., General Electric NAP-5).
- 0.22μm centrifugal filter units (e.g., Merck Millipore Ultra-
free®-MC, GV 0.22μm). - Reverse transcription (RT) kit (e.g., Toyobo ReverTra Ace®
qPCR RT Kit).
31.EcoRI,BamHI,BglII, andHindIII restriction enzymes. - DNA Ligation Kit (e.g., Takara Bio
). - Competent high DH5αE. Coli(e.g., Toyobo Co., Ltd.).
- Miniprep Kit (e.g., QIAGEN QIAprep Spin Miniprep).
- Midiprep Kit (e.g., QIAGEN HiSpeed Plasmid Midi).
- 4% PFA in 0.1 M PBS (e.g., Muto Pure Chemicals).
- pUC19 DNA (e.g., Takara Bio).
- pSuper.neo vector (Oligoengine).
- LB plate containing 100μg/mL of carbenicillin.
- 1PBS.
- Hoechst 33342 (e.g., Dojindo Molecular Technologies).
2.2 Oligonucleotides 1. N60 template DNA: 5^0 -GAA TTC CGC GTG TGC ACA
CC–N60–GTC CGT TGG GAT CCT CAT GG-3^0.
- Forward primer: 5^0 -GCT AAT ACG ACT CAC TAT AGG
GAA TTC CGC GTG TGC ACA CC-3^0 (T7 promoter
sequence is underlined). - Reverse primer: 5^0 -CCA TGA GGA TCC GAA CGG AG-3^0.
- RT aptamer template 1: 5^0 -GAT CCC CGG AGC AAT GAT
GGCCTA GAT AAA TTC GGAGCT TGA TCT TCA TTT
TTA-3^0 (RNA recognition arms are underlined, and the
BHQ1-binding loop is bold). - RT aptamer template 2: 5^0 -AGC TTA AAA ATG AAG ATC
AAG CTC CGA ATT TAT CTA GGC CAT CAT TGC TCC
GGG -3^0.
Live-Cell RNA Imaging with a Small Molecule 307