"6-Phenylpyrrolocytosine (PhpC) is a cytosine mimic with excellent base-pairing fidelity, thermal stability, and high fluorescence." This base modification was introduced into siRNAs, and did not affect stability or gene silencing activity. These molecules were used to track and monitor uptake and trafficking of siRNAs within cells in real time. I found it really interesting that this modification did not alter activity of the siRNA molecule - though the base pairing is the same i thought the addition of a large bulky ring in the major groove would interfere with duplex binding. Innate immunostimulation was also repressed, which can be a big draw back for other nucleotide modifications (such as fluorination, a previously hot blog topic). Expanding the types of modifications introduced into nucleotides that are strong biological mimics will help advance research using fluorescent nucleic acid molecules.
This is cool! I am just wondering what the advantage is of having a fluorescent base instead of labeling the 3' or 5' end of the siRNA with a fluorophore?
ReplyDeleteThe fluorescence of modified bases if often quenched through base pairing interactions, allowing researchers to use these as tools to specifically study binding events of the siRNA molecule. Spectral changes can be used to determine when/where these molecules are binding to complimentary DNA or RNA, as well as on/off kinetics, which is an advantage when compared to 3' or 5' labels.
ReplyDeleteThat's what I originally thought too, but that's not true for this paper, right? No changes in fluorescence here upon hybridization.
ReplyDeleteDo you have a reference for examples of fluorescent nucleobases being used to detect oligonucleotide duplex formation (through either increase or decrease of fluorescence)?
It is true here - figure 2 shows that the duplex signal is typically less than the single stranded precursors. There are clearly fluorescent changes during hybridization. The overhang modifications are the only aberrations to this trend. They didn't specifically investigate quenching however, since they were looking at # of PhpC molecules needed. They state "The fluorescence of PhpC-containing oligonucleotides is generally quenched when hybridized to complementary RNA or DNA."
ReplyDeleteHere are references (taken directly from this paper):
1)http://nass.oxfordjournals.org/content/52/1/399.full.pdf
2)http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2817455/?tool=pubmed
1 shows trend of quenching with double stranded formation, 2 shows use to determine RNAseH activity similar to molecular beacons.
In some cases in RNAi, one strand is quickly degraded while the other persists in the cell. By doing the experiment twice, with either the sense or antisense tagged, you could also see if one strand is being degraded.
ReplyDeleteAlso, if you give a creature phenylpyrrolocytosine-triphosphate, would it incorporate it into its own RNA?
You are right James! I just missed the quenching, because it is so weak (only 50% reduction in the best case) and I was looking for an application of that effect (in a micrograph). I guess there is still plenty of room for improvement.
ReplyDelete