3 Methods
3.1 Probe Design 1. Choose the target sequence (preferentially 18–20 nucleotides
in length) you want to detect in cells.
- Check the number of copies, structure, singularity, and the
specificity of the target. - For nuclease resistant probes the combined use of 20 O-Me
RNA, DNA, and LNA (XL) is required. - The optimal position of the dye has to be determined by
replacing different nucleotides by the Ser(TO/QB)-monomer
(seeNote 1). - Afterwards all probes have to be measured under the same
conditions to compare the fluorescence properties.
3.2 Probe Synthesis All phosphoramidites are used according to manufactures
instructions.
- After synthesis the CPGs are dried under reduced pressure.
- The CPG is deprotected in 1 mL of aqueous ammonia (32%)
for 2.5 h at 55C. - After centrifugation the supernatant is collected.
- The volatiles are removed at reduced pressure and the residues
are dissolved in water. - The crude product is purified DMTr-on by preparative RP-
HPLC. - Afterwards, the DMTr group is removed upon treatment with
300 μL of 80% aqueous AcOH for 15 min at room
temperature. - The detritylation mixture is treated with 1 mLiPrOH and the
resulting precipitate again purified by RP-HPLC DMTr-off. - Finally, the oligonucleotides are desalted by precipitation with
3 M ammonium acetate (10% vol.) and 1 mLiPrOH. The
pellets are dissolved in water (Millipore) and analyzed.
3.3 Probe
Characterization
- The amount of substance is determined using Lambert–Beer
law, the absorption at 260 nm and the calculated extinction
coefficient e.g.,https://eu.idtdna.com/calc/analyzer. - The purity is identified by using analytical RP-HPLC-UV
(Absorption: 260 nm) with 1 nmol of probe solved in water. - Characterization by MALDI-TOF mass spectrometry requires
approx. 1 nmol of probe in HPA matrix for measurements in
the positive detection mode.
278 Jasmine Chamiolo et al.