This protocol is for conjugating NHS-ester modifications, such as TxRd (Sulforhodamine 101-X) (protocol is effective for most NHS-ester modifications), to an oligonucleotide with an amino label, such as 5'-Amino-Modifier C6 (Figure 1). Since TxRd (Sulforhodamine 101-X) and several other modifications are only available as NHS esters, they must be manually conjugated to amino-labeled oligonucleotides in a separate, post-synthesis reaction.
The conjugation process is divided into two main steps: 1) the conjugation reaction, and 2) the removal of excess, free NHS-ester modification by post-conjugation precipitation.
Though HPLC is the conventional method of removing excess, free NHS-ester modification and uncoupled oligonucleotide, other purification processes, e.g. precipitation, may be effective too. Precipitation (described below) removes most but not all remaining NHS-ester modification. Depending on the application, the unconjugated modification may be a problem. While it will not react further since the NHS-ester is hydrolyzed by the buffer, it may contribute to background noise and therefore lead to a poor S:N ratio (if the modification is a dye, e.g. TxRd (Sulforhodamine 101-X) or reduce surface-loading density (if the modification serves to attach, e.g. biotin). In addition, uncoupled oligonucleotide will remain.
Start with the recommended conditions and optimize as needed.
The post-conjugation precipitation is performed in a 2 mL tube (50 mL tube for a larger reaction scale). Be sure to protect dyes from photobleaching.
The oligonucleotide is now conjugated to the modification and may be dissolved in water or buffer for use in the intended application.
Preparation of 50 mL of 0.091 M NaB Buffer
Other non-amine buffers may be substituted for NaB as long as the pH is no higher than 8.5 (a higher pH will hydrolyze the NHS-ester too quickly).
Preparation of NHS-Ester Modifications
NHS-ester modifications come as anhydrous reagents supplied in bottles with airtight bottle-cap seals. Keep moisture away from NHS-esters prior to addition to the amino-labeled oligonucleotide buffer solution as they hydrolyze quickly. Use anhydrous DMSO or another solvent.