Table 2. Conjugation strategies for dyes or biotin.
| Chemistry/Method | Reactive Group | Remark |
|---|---|---|
| DNA/RNA | ||
| Phosphoramidite or acetoxyethoxy methyl (ACE) solid support synthesis | Direct incorporation into the backbone | |
| Amine reactive (-NH2) | succinimidyl (NHS) ester | Amino C6- dT/dC (for internal labeling without backbone disruption) |
| Thiol reactive (-SH) | Maleimide | Thiol modifier on 3′or 5′ end |
| Proteins | ||
| Amine reactive (-NH2) | succinimidyl (NHS) ester | N-terminal or Lysine amine groupa |
| Thiol reactive (-SH) | Maleimide | Cysteine thiol groupb |
| Ketone reactive (=CO) | Hydrazine | Unnatural amino acid with ketone groupc |
a
Achieving high selectivity and specificity with amine labeling is usually complex in proteins since they naturally carry multiple lysine residues.
b
Double cysteine mutant proteins can also be labeled with equimolar ratios of donor and acceptor dyes simultaneously where molecules carrying the right dye pair can be identified by their FRET signature79. Other double labeling strategies have also been attempted for better specificity75, 95, 96.
c
Incorporation of the ketone group using an orthogonal tRNA-synthase pair that exclusively recognizes the amber codon can be used36.