Protocol for creating 5′-Phosphorylated Guides from Unmodified Oligonucleotides for use with TtAgo (NEB #M0665)

5′-phosphorylated guide may be ordered directly from a supplier (e.g., Integrated DNA Technologies, Inc.), however, be sure to verify that the phosphate modification is present by reviewing the QC data for the oligo. This will typically be in the form of ESI MS showing the oligo is the correct/expected mass which includes the phosphate modification.

Alternatively, 5′-phosphorylated guide may be prepared by treating an unmodified oligo with kinase. This can be a more cost-effective option when a large number of guides needs to be prepared. 5′-phosphorylated guides may be prepared from unmodified DNA oligonucleotides using one of the following procedures:

Small-scale: routine phosphorylation of individual guide oligonucleotides

  1. Set up a reaction for each unmodified oligonucleotide to be used as a guide with argonaute. This can be done in PCR tube strips for convenience. For 100 µl reactions yielding 5 µM 5′-phosphorylated guide, set up the following in the order provided:

    Nuclease-free water (NEB #B1500) 84 µl
    T4 DNA Ligase Reaction Buffer (10X) (NEB #B0202) 10 µl
    DNA Oligonucleotide (100 µM)
    (16-18 nt)
    5 µl
    T4 Polynucleotide Kinase (T4 PNK) (10 U/µl) (NEB #M0201) 1 µl

    Note that reactions can be scaled up or down multiplicatively depending on how much guide is needed.

    T4 DNA Ligase Reaction Buffer can be used in place of the T4 Polynucleotide Kinase Reaction Buffer reaction buffer as this is a non-radioactive labeling procedure, thus eliminating the need to add ATP as a separate component to the reaction. More information can be found here.

  2. Incubate reactions at 37°C (phosphorylation), followed by, 65°C for 20 min (heat-inactivate/denature T4 PNK).

  3. Denatured T4 PNK does not interfere with the argonaute endonuclease reaction, and the guides are ready to use. However, if purification of the phosphorylated oligo from the denatured PNK is desired, the Monarch® PCR & DNA Cleanup Kit (NEB #T1030) may be used following the modified Oligonucleotide Cleanup protocol.

  4. Quantify purified phosphorylated oligonucleotides by UV absorbance at 260 and 280 nm before use.

  5. Phosphorylated guides may be stored at 4°C for up to two weeks. If stored frozen at -20°C phosphorylated guides exhibit the same level of activity for several months to a year.