RNA Detection

6. Remove the supernatant and rinse the pellet with 70% ethanol.


7. Air-dry the pellet for 5 min, and dissolve it in a 15-μL TE buffer
   solution.

3.3.4 Reverse
Transcription

1. Heat the eluted resin-bound RNA for 5 min at 65C, then
   immediately chill it on ice.


2. Prepare the RT reaction mixture.


3. The reaction is performed for 15 min at 37C, followed by
   heat-inactivation of RTase at 98C for 5 min.


4. 4.6μL of the reverse-transcribed products are used as templates
   for PCR amplification to generate the DNA pool for the next
   round of selection (seeNote 12).

3.3.5 Cloning and
Sequencing of RNA
Aptamers for BHQ1

1. After the final round of selection, digest the resulting DNA
   pool with restriction enzymesEcoRI andBamHI.


2. Prepare the ligation mixture withEcoRI andBamHI pUC19
   plasmid as a vector.


3. Incubate the mixture for 30 min at 16C to allow the ligation
   reaction.


4. Transform DH5αwith the ligation product, and heat shock at
   42 C for 1 min and spread the cells on an LB plate containing
   100 μg/mL of carbenicillin.


5. Incubate the plate overnight at 37C.


6. Select a single colony and culture overnight at 37C in a liquid
   LB medium, and isolate the plasmid using the QIAprep Spin
   Miniprep Kit.


7. Sequence the insert DNA using primers in the vector backbone
   (seeNote 13).

3.4 Preparation of
RNA-Targeting
Aptamer (RT-Aptamer)

The specific RNA-recognition aptamer is prepared as shown in

Fig.2.

1. The stem region is removed from the BHQ1-binding stem-
   loop structure [20], leading to the formation of a flexible
   BHQ1-binding loop that lacks in BHQ1-binding activity.


2. Two short RNA sequences (RNA targeting arms),
   complementary to a 24-base oligonucleotide in a target
   RNA sequence, are attached to the flexible structure (see
   Note 14).


3. The RNA-targeting arms hybridize to a target RNA, conse-
   quently forming the BHQ1-binding loop on the target mRNA
   (seeNote 15).

Live-Cell RNA Imaging with a Small Molecule 313