A successful PICh experiment requires the use of special probes which bind to the locus of interest with high stability and specificity. PICh probes are not commercially available, or may require custom synthesis by Exiqon, the inventor and owner of the LNA (Locked Nucleic acid) patent. Conventional DNA based FISH probes, such as the ones obtained by nick translation, may not be suitable for PICh, as we think (and know) their binding to the target is not stable enough to maintain sufficient material bound during the course of the purification. PICh probes typically contain a mixture of DNA and LNA residues, a very long spacer and desthiobiotin. The positions and content of LNA residues in the probe do not obey specific rules, however we are always designing our probes as follows:
-40-50% LNA content scattered along the length of the sequence (no ‘blocks of LNA’)
-25-35 nucleotides long with a Tm generally higher than 78°C.
Following these rules is not a guarantee PICh will work, since other critical parameters contribute to a successful purification. This design is however the best we found so far.
In the original paper, we were using a 108 atoms long spacer together with desthiobiotin. We are now using a different strategy, which works with the same efficiency: we are coupling 4 times a Spacer 18 and a Desthibiotin Tri-Ethylene-Glycol, making a ~100atoms long distance between the DSB and the oligo. These extremely long spacers are used to prevent steric hindrance issues that could potentially be encountered upon immobilization of chromatin on beads.
Here we provide a synthesis protocol for making PICh probes in the lab using conventional nucleic acid synthesis methods.
Modern nucleic acid synthesizers allow using different types of chemistries, but phosphoramidite chemistry is the most popular and the easiest one to achieve efficient synthesis. Phosphoramidite monomers are stable and allow generating specific ribo- and deoxy-ribo oligonucleotides with almost any kind of modification of interest, provided they are available as amidites precursors (fluorescent label, small tags, spacers, etc…). The
nucleotide chain grows from an initial protected nucleotide attached via its 3’ end to a solid glass support (Controlled Pore Glass or CPG) in a column. Chemical synthesis of oligonucleotides always runs from the 3’ toward the 5’ end of the oligo.
Similar to a PCR reaction where each cycle is divided into Annealing/Polymerization/Denaturation steps, oligo synthesis is a step-wise chemical reaction where an amidite is added 5’ of the previous nucleotide. In these reactions, spacers and biotin are considered similar to nucleotide since they are also used as phosphoramidites precursors. To obtain desired oligonucleotides, monomers are added following a stepwise chemical reaction called a cycle. A synthesis cycle consists of 5 steps (figure 1):
- Deblocking: The cycle begins with the removal of the acid-labile 4,4’ DimethoxyTrityl
(DMT) group from the 5’ hydroxyl end.
- Coupling: The next protected phosphoramidite (with a 5’ DMT) is delivered to the reaction column in the presence of tetrazole which activates the coupling reaction.
- Capping: Unreacted 5’ hydroxyl ends, not engaged in a reaction with the incoming monomer must be permanently blocked. This step is necessary to minimize truncated products, to increase the synthesis yield and to facilitate the purification process. Tetrahydrofuran (THF) with Methylimidazole and Pyridine are used to acetylate and block the unextended 5’hydroxyls.
- Oxidation: Phosphite triester are oxidized to yield the phosphor diester bond linking the two nucleosides.The cycle of monomer addition is then completed and other cycle begins with the removal of the 5’DMT from the previously added monomer.
- Capping: An extra capping step is necessary to ensure no hydroxyl group is left.
We are using The Expedite 8909 Nucleic Acid Synthesis System (Note1) operated by a PC computer running the “Expedite Workstation” software version 2.5, 1999 from Perseptive Biosystems. This system, although old and not sold anymore as a brand new instrument, can be obtained as refurbished equipment from several synthesizer companies. The 8909 has very low reagent consumption, making this a cost effective machine for PICh probe synthesis.
Two columns can operate independently allowing the simultaneous synthesis of two different sequences. PICh probes may also be made using other systems. Please refer to the manufacturer’s instruction for adapting the protocol below to other machines.
Institute of Human Genetics. CNRS UPR1142 / 141 rue de la Cardonille. 34000 MONTPELLIER FRANCE
Corresponding author: Elodie Rey