I am trying to design 20 bp ssDNA oligos in order to strongly bind mRNAs with high specificity at room temperature or colder in high cation concentration (1 M). I'd like to avoid the formation of hairpin loops as much as possible. So far I've come up with the following:
- medium GC content (40-50%). ssDNA-mRNA binding needs to be strong, but too high GC = hairpin loops
- perhaps heat the mixture to 35C to melt hairpins and cool down to RT to reanneal to mRNAs?
Would anybody else have additional tips or changes to the ones above?
Many thanks
First before you order your 20 bp ssDNA oligo, run your sequence through Oligo Analyzer: http://www.idtdna.com/analyzer/applications/oligoanalyzer/
Check for hairpin formation, self-dimer and hetero-dimer formation.
The software gives you pretty good insight into your oligo sequence.
First before you order your 20 bp ssDNA oligo, run your sequence through Oligo Analyzer: http://www.idtdna.com/analyzer/applications/oligoanalyzer/
Check for hairpin formation, self-dimer and hetero-dimer formation.
The software gives you pretty good insight into your oligo sequence.
High salt and slow annealing are good, as they will promote duplex over hairpin formation. High concentration would be another duplex promoting condition. I would heat to much higher T than 35 though before annealing, although Im not sure what you are trying to do - maybe you want the mRNA in as 'native' conformation as possible? If you are looking at sequence specific binders, you should also predict the possible basepairing within the mRNA to see if there is competition with the mRNA fold.
Is there any specific reasonthat forces you to go down in the annealing temperature? you can get modified oligonucleotides as hybridization probes with higher Tm and target affinity giving you way better mismatch discrimination.
I agree with previous comments (to check first the oligos). I will suggest also that you increase (if it is possible) the T. The best way to get a good binding is to denature both (mRNA and ssDNA oligos) together in an incubation bath with water and to let cool down slowly. Another possibility is to make the same procedure using a PCR apparatus by setting a slow ramp down (5°C/min could be a good option).
You need to take in consideration that Magnesium and other metals catalyze non-specific cleavages in RNA. This kind of cleavage is increased with the increase in temperature, so you need to avoid the presence of such elements when you are performing the denaturing step.
If you need to denature the ssDNA oligos separately from the mRNA I suggest that you incubate the oligos at 95°C during 2 min and snap cool on ice before you mix them with the mRNA.
for ssDNA incubation at 95 degree for 2 min is good suggestion, but for mRNA no more than 75C for 2min then cool immediately on ice.
Many thanks for the tips. Unfortunately I cannot go higher than 35C without damaging the sample. EDTA in the buffer should prevent non-specific RNA cleavages. I've used mFold to predict mRNA regions of self-hybridisation and have designed oligos that target single-stranded regions. Using OligoCalc I've found that deltaG for the hybridisation is approx 100 times higher than the formation of the hairpin loop. Would this be an indicator of a much stronger affinity for oligo hybridisation than folding?
If your oligo is just 20 nt long and you are working around RT, it will pretty much end up in its thermodynamically most stable form whatever elaborate annealing protocol (temp) you use. If your oligo is not self complementary just design the sequence that it cannot form a stable hairpin or partial duplex. With a 20 nt DNA the most stable HP would be ~8 base pairs and 4 nt in a loop. What you could do is to put A's at the center of the sequence. Hairpin loops with As are less stable than T's for example.
As aways low concentration and low salt will favor hairpin over duplex. If you are worried, just run a native PAGE on your oligo at different concentrations. (either 32P labeled) or with fluorescence detection, hairpins have very different mobilities than a ss DNA. So of if your oligo runs like T20 or so on a gel under similar conditions (temp buffer as in your assay, you are fine)
BTW a QUICK Heat chill cycle 95 deg -> ice, will encourage formation of hairpins (if the sequence allows that... (intramolecular hairpin formation is quick... the partner strands do not have to meet each other) What it will do is break up aggregates, dimers and multimers (if possible) temporarily.
Another idea is to design your oligo as a LNA/DNA hybrid. Oligonucleotides containing LNA are generally recommended for use in any hybridization assay that requires high specificity and reproducibility. In addition, LNAs offer the possibility to adjust Tm values of primers and probes in multiplex assays.
If you assay requires high specificity, design several oligo with different region of mRNA, when you run your experiment, in the hybridiization step probably need to do differents condition with cation concentration.
Many thanks, folks. I'll redesign my oligos with some of these tips in mind. Running a gel with the oligos is indeed a good idea to rule out hairpin loops as an inhibitory factor if the results are poor. I've considered the LNA/DNA hybrid, but also morpholinos. Both, I believe, have similar properties for my application (high specificity, nuclease resistance (better with morpholinos), strong binding). Any reasons I'm missing why either LNAs or morpholinos stand out as better candidates?
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