This modification is a post synthesis NHS conjugation to a primary amino group thus an additional modification with an amino group is required. A C6 or C12 amino group can be placed at the 5' or for the 3' end a C3 or C7 amino and for internal positions an amino modified base is used, e.g Amino dT C6.
YIELD
NHS based modifications are post synthesis conjugation performed using a primary amino group. The yield is lower as compared to direct automated coupling of modifications that are available as amidites. Approximate yield for various scales are given below.
~2 nmol final yield for 50 nmol scale synthesis.
~5 nmol final yield for 200 nmol scale synthesis.
~16 nmol final yield for 1 umol scale synthesis.
Click here for a complete list of Click Chemistry Oligo Modifications
Photocleavable DBCO-NHS ester contains a spacer arm containing a photocleavable moiety, this can be efficiently photoreleased, typically >90% in 5-25 minutes using an near-UV low intensity lamp (e.g. 365 nm lamp at 1-5 mW/cm2).
Cyclooctyne-based modifications offers the ease of copper-free click reagents. These are simple to use and has excellenet click performance in 17 hours or less at room temperature. Gene Link offers 5'-DBCO-TEG for preparing oligos with 5'-DBCO and a 15 tom triethylene glycol spacer arm, DBCO-dT for inserting a DBCO group at any position within the oligonucleotide and DBCO-sulfo-NHS Ester is also offered for post-synthesis conjugation reactions. DBCO-modified oligos may be conjugated with azides in organic solvents, such as DMSO, or aqeous buffers. Depending on the azide used, the reaction will go to completion in 4-17 hours at room temperature.
Photo Cleavage Protocol
Cleavage occurs by irradiation with near-UV light (300-350 nm, >90% cleavage occurs within 5-25 minutes. Try using a Black Ray XX-15 UV lamp (Ultraviolet Products Inc., San Gabriel, CA) at a distance of 15 cm (emission peak 365 nm, 300 nm cut-off, 1.1 mW intensity at~31 cm).
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