RNA Detection

7. To conjugate the fluorophore, apply 3.0μmol Cy3-NHS-ester
   in 36μL DMSO to the resin, and incubate it overnight at room
   temperature in the dark to obtain Compound 2.


8. The resin is then washed with DMF and dichloromethane (see
   Note 6).


9. To cap the unreacted amino groups on the resin, the resin is
   treated with 0.5 mL 25% acetic anhydride in dichloromethane
   at room temperature for 5 min.


10. Wash the resin with dichloromethane three times for 1 min.


11. To cleave Compound 3 from the resin, TFA–triisopropylsila-
    ne–H 2 O (95:2.5:2.5) is added to the resin and incubated at
    room temperature for 2 h.


12. The mixture is then filtered and concentrated in vacuo, and the
    residue is lyophilized with H 2 O.


13. To couple with BHQ1, the residue is dissolved in a solution of
    3.0μmol HCTU and 3.0μmol HOBt in 20μL DMSO, and is
    incubated at room temperature for 10 min.


14. Subsequently, 7.9 μmol N,N-diisopropylethylamine and
    3.0μmol BHQ1 amine are added to the dissolved residue,
    and the mixture is incubated overnight at room temperature
    in the dark to obtain Compound 4 (BHQ1-Cy3 probe) (see
    Note 7).

O

O

NHFmoc O

O

N

H

O

O

NHFmoc

O

O

N

H

O

O

H

N

O

4

4

HO

O

N

H

O

O

H

N

O

4

N

H

O

N

H

O

O

H

N

O

4

β-Ala-wang resin

step 1

BHQ1 Cy3

Cy3

Cy3

1

2

3

4 (BHQ1-Cy3)

step 2

step 3 step 4 step 5

step 6 step 7 step 8

Scheme 1Synthesis of BHQ1-Cy3 probe

310 Shin-ichi Sato et al.