Glen Report 21.2: Deprotection - Volume 3 - Dye-Containing Oligonucleotides

Volume 3: Dyes - Deprotect to Completion

  1. Even with oligos containing sensitive dyes, the nucleobases must be fully deprotected for full functionality.
  2. Will the dye-labelled oligo survive my preferred deprotection scheme?
  3. If not, which deprotection scheme will fit best with my equipment and purification strategy?

In the previous two articles in this series on Deprotection, we focused on DNA and RNA deprotection. Our first priority in deprotection is to "Deprotect to Completion" by removing 100% of the protecting groups on the nucleobases, while following the mandate to "Do No Harm". Dyes tend to have the unfortunate properties of being quite sensitive to the basic conditions of oligonucleotide deprotection while being expensive. The "Do No Harm" stricture is doubly important when deprotecting dye-labelled oligonucleotides. To make matters worse, many dye labelled oligonucleotides also contain a quencher molecule that may also be base sensitive. This combination of properties is guaranteed to lead to confusion and possibly decomposed, worthless oligos may result if incompatible deprotection conditions are used.

In this article, we have generated a Table which we hope will remove some of the challenges from the deprotection of dye-labelled oligonucleotides. We have focused on a variety of methods for oligonucleotide deprotection:

  1. 30% NH4OH 17 hours at 55 °C; sufficient to deprotect all standard bases, A/C/G/T
  2. 30% NH4OH 17 hours at room temperature; sufficient to deprotect A, C and dmf-dG
  3. 30% NH4OH 2 hours at 65 °C; sufficient to deprotect A, C and dmf-dG
  4. 30% NH4OH 2 hours at room temperature; sufficient to deprotect only UltraMild monomers, Pac-dA, Ac-dC, ipr-Pac-dG when UltraMild Cap A is used.
  5. 50 mM Potassium Carbonate in Methanol for 4 hours at room temperature; sufficient to deprotect only UltraMild monomers, Pac-dA, Ac-dC, ipr-Pac-dG when UltraMild Cap A is used.
  6. Tert-Butylamine/water 1:3 (v/v) 6 hours at 60 °C; sufficient to deprotect A, C and dmf-dG.
  7. 30% Ammonium Hydroxide/40% Methylamine 1:1 (v/v) 10 minutes at 65 °C; sufficient to deprotect all standard bases, however, Ac-dC must be used.

The Table illustrates the conditions suitable for deprotecting oligos containing one or two of the dyes listed. We will continue to update this Table on our web site.

Note: JOE has not been tested with Condition F.

$ Denotes an acceptable, but not preferred method.

Table: Deprotection Conditions Suitable for Popular Dyes and Quenchers
FAM TET HEX JOE TAMRA Yakima
Yellow
Cy3/
DyLight 547
Cy5/
DyLight 647
Dabcyl Eclipse BHQ-1 BHQ-2 BHQ-3
FAM A..G A..G B, D, E,G A..E, G E, F A..D, F B..E, G D, E A..E, G A..G $A, B..E, $F,G A..E $B, D
TET A..G A..G B, D, E, G A..E, G E, F A..D, F B..E, G D, E A..E, G A..G $A, B..E, $F,G A..E $B, D
HEX B, D, E, G B, D, E, G B, D, E, G B, D, E, G E B, D B, D, E, G D, E B, D, E, G B, D, E, G B, D, E, G B, D, E $B, D
JOE A..E, G A..E, G B, D, E, A..E, G E A..D B..E, G D, E A..E, G A..E, G $A, B..D A..E $B, D
TAMRA E, F E, F E E E, F F E E E E, F E, $F E Incompatible
Yakima
Yellow
A..D, F A..D, F B, D A..D F A..D, F B, C, D D A..D A..D, F $A, B, C, D, $F A..D $B, D
Cy3/
DyLight 547
B..E, G B..E, G B, D, E, G B..E, G E B, C, D B..E, G D, E B..E, G B..E, G B..E, G B..E $B, D
Cy5/
DyLight 647
D, E D, E D, E D, E E D D, E D, E D, E D, E D, E D, E D
Dabcyl A..E, G A..E, G B, D, E, G A..E, G E A..D B..E, G D, E A..E, G A..E, G $A, B..E, G A..E $B, D
Eclipse A..G A..G B, D, E, G A..E, G E, F A..D, F B..E, G D, E A..E, G A..G $A, B..E, $F,G A..E $B, D
BHQ-1 $A, B..E, $F,G $A, B..E, $F,G B, D, E, G $A, B, C, D E, $F $A, B, C, D, $F B..E, G D, E $A, B..E, G $A, B..E, $F,G $A, B..E, $F,G $A, B..E $B, D
BHQ-2 A..E A..E B, D, E A..E E A..D B..E D, E A..E A..E $A, B..E A..E $B, D
BHQ-3 $B, D $B, D $B, D $B, D Incompatible $B, D $B, D D $B, D $B, D $B, D $B, D $B, D