Conjugates of Uridine and GFP-like Chromophore for Detection of Genetic Material

Abed Saady abed.saady@biu.ac.il Bilha Fischer
Chemistry, Bar Ilan University, Ramat Gan, Israel

We recently showed that dUTO, 1, in an oligonucleotide probe targeting cyclin D1 mRNA (a breast cancer marker), in total RNA extract from cancerous cells, allowed the sensitive detection of the target based on a 7-fold enhanced fluorescence of the probe.1 Yet, a useful fluorescent probe for living cell imaging has to have an excellent signal/background ratio. Therefore, here, we aimed to prepare single stranded 2’-OMe-RNA probes which covalently bind, via a suitable spacer, a duplex-intercalating moiety which has an extremely low Φ in water, and which upon probe hybridization with target mRNA will exhibit significant Φ and brightness. Specifically, we developed novel uridine analogues bearing a GFP-like chromophore as an intercalator, 2, for incorporation into an oligonucleotide probe, to be used for the detection of breast cancer markers, HER-2. We successfully synthesized analogues of the GFP-chromophore, 2 and then conjugated them to uridine via a 4-atom nitrogen-containing spacer to get compound 3. Nucleotide conjugate 3 was prepared from 5-(3″-Aminoallyl)-2′-deoxyuridine, which in turn was obtained by Heck reaction between 5-iodo-2`-deoxyuridine and N-allyltrifluoroacetamide in 57% yield. We then removed the trifluoroacetamide protecting group by hydrolysis in ammonium hydroxide in 99% yield. 5-(3″-Aminoallyl)-2′-deoxyuridine was coupled with 2, to give 3 in 47% yield. Finally, the corresponding monomer for oligonucleotide synthesis was prepared by 5’-OH DMT protection and activation of 3’-OH with phosphoroamidite, 55% yield. Chromophore 2 both in methanol and glycerol exhibited λabs 381 (nm) and ϵ (M-1 cm-1) 20600. Addition of two ortho flourine atoms in the phenol provided compound 4, improving the absorption values: λabs (nm) and ϵ (M-1 cm-1) to 386 and 25600, respectively. Compound 3 did not fluoresce in methanol; however, in glycerol, which is 3000 times more viscous than methanol, emission was observed at 460 nm (Φ= 0.31, Brightness= 11.7). This finding supports our hypothesis that a rigid medium (e.g. base-pairs of an RNA duplex) limits the free-rotation of the intercalator, resulting in a fluorescent signal.









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