RNA Detection

15. The mixture is diluted and acidified with 0.6 mL of 10 v/v %
    TFA in acetonitrile–H 2 O (1:1).


16. The mixture is then purified by HPLC (0–3 min: CH 3 CN,
    60%; 3–23 min: CH 3 CN, 60–80% at RT,tR¼19.2 min).
    Compound 4 (BHQ1-Cy3 probe) is obtained as a dark purple
    amorphous solid (1.5 mg, 82% based on the resin) by lyophili-
    zation (seeNote 8).


17. The BHQ1-Cy3 probe is dissolved in DMSO and stored at
     30 C.

3.3 In Vitro Selection
of RNA Aptamers

In vitro selection is performed according to standard procedure for

isolating RNA aptamers for BHQ1 [22–24] (Fig.1). The selection

cycle is repeated several times to enrich RNA species specifically

bound to BHQ1.

3.3.1 Preparation of DNA
Pools (SeeNote 9)

1. Prepare the PCR mixture in PCR tubes.


2. Place the tubes in a thermal cycler and perform the PCR
   amplification using the following program: 2-min initial dena-
   turation at 94 C, followed by 30 sequential cycles of

NH O NH

O O

N

NN

NN

NO 2

OMe

NH O NH

O O

N

NN

NN

NO 2

OMe

T7 promoter

Randomized

sequence (N60)

DNA pool

Unbound RNA

cDNA pool

DNA pool RNA pool

Binding to compound

immobilized resin

Transcription by T7

RNA polymerase

Regeneration by

transcription

Amplification

by PCR

Reverse

transcription

Washing

Elution

Fig. 1In vitro selection of BHQ1 aptamers

Live-Cell RNA Imaging with a Small Molecule 311