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  • br Fig Target NIC dUHBI analog due


    Fig. 4. Target NIC-dUHBI analog.  due to restricted rotation, and therefore high F. In this way, enhanced fluorescence is indicative of the presence of target mRNA (Fig. 3).
    dU was selected as the nucleoside for conjugation with HBI-analog due to its relatively easy application to various synthetic reactions; the dU nucleobase requires no protective groups, and has a reactive C5-position suitable for chemical elaboration. Further-more, natural H-bonding base-pairing and standard solution conformation of dU are not disturbed due to C5-substituents [18].
    Specifically, the scaffold of the (5-(4-methoxybenzylidene)-3-methyl-2-thioxo-4-imidazolidinone) chromophore, 5, was synthe-sized in two steps starting from 3-methyl-2-thioxoimidazolidin-4-one, 6 [19]. The latter was treated with p-methoxybenzaldehyde 7 [20], to give 5 by Aldol condensation (82% yield) [21] (Scheme 1).
    Compound 4 was obtained in 90% yield upon treatment of 10 with 3% trichloroacetic Butyrolactone I in dichloromethane, and was used to measure the photophysical properties of 20-dUHBI. Finally, the 20-
    phosphoramidite monomer 11, which was required for
    oligonucleotide synthesis, was obtained in 74% yield upon treat-ment of 10 with 2-cyanoethyl N,N,N0,N'-tetraisopropylphosphor-odiamidite and 4,5-dicyanoimidazole (DCI) (Scheme 4). The 31P NMR spectrum showed two typical phosphoramidite signals at 162.20 ppm and 161.62 ppm, respectively, for the two di-astereoisomers resulting from the stereogenic phosphorous atom. Monomer 11 enabled the facile incorporation of 20-dUHBI into oligonucleotide ON1 (50-CCCGTUTCAACAGGAGTTTC-30) at an in-ternal position via oligonucleotide synthesis under usual conditions.
    prepared 5-(4-methoxybenzylidene)-3-methyl-2-thioxo-4-imidazolidinone, 5 [17], to be utilized as an intercalator. This HBI fluorophore was conjugated via a 7-atoms spacer to 20-dU at its C-5 position (thus not interfering with natural H-bond base-paring). dUHBI conjugate 4 was incorporated into a DNA sequence target-ing HER2 mRNA.
    Compound 4 results from the conjugation of HBI chromophore 5 (Scheme 1), and 20-deoxyuridine via a 7-atom spacer. The 50- and 30-OH positions of the ribose are available for protection by 4,40-dimethoxytrityl chloride (DMT-Cl) and activation as a phosphor-amidite, respectively, both of which are required for incorporation of nucleoside 4 in an oligonucleotide probe. The length of the flexible spacer was chosen as 7 atoms, long enough to allow for intercalation of the chromophore in the hybridized probe:target mRNA duplex. Intercalation results in reduced non-radiative decay
     2.3. Photophysical characterization of dUHBI, 4
    In order to provide an initial indication of the fluorescence that we expected to detect upon hybridization of the dUHBI-oligonu-cleotide probe with target mRNA, we established the photophysical properties of nucleoside 4 prior to its incorporation into an oligo-nucleotide. The photophysical properties of compound 4 were evaluated in various solvents at a range of viscosities (Table 1). UV spectra of compound 4 in all solvents exhibited labs and ε around 387 nm and 20600 M 1cm 1, respectively.
    Next, we measured the quantum yield of 4 in various solvents and at a range of glycerol: methanol ratios, representing a range of viscosities (from 0.54 to 934 cp) (Table 1). The fluorescence of compound 4 was viscosity-dependent and increased in viscous solvents; for example, compound 4 barely fluoresces in methanol (F 2.91 10 3); however, in glycerol, which is ca. 3000 times more
    Scheme 1. Reaction conditions: piperidine, ethanol, reflux, 24 h, 82% yield.
    Notably, in 1,3-butanediol and in glycerol: methanol (7:3) mixture, which share the same viscosity, F of 4 is 0.019 and 0.014, respectively, thus implying that viscosity is a major parameter 
    affecting the emission of HBI analogues. This finding supports our hypothesis that rigid medium limits the free-rotation around the methine-bridge of the HBI analog, thus resulting in a fluorescent signal, and hence, high fluorescence is expected upon intercalation of the HBI-moiety.
    Table 1
    Photophysical data of compound 4 in various solvents at a range of viscosities.
    Viscosity (cp)
    2.4. Selection of the sequence of the hybridization probe
    To test the applicability of the dUHBI-labeled oligonucleotide probe for the detection of HER-2 mRNA, we selected a 20-mer sequence (50-CCCGTUTCAACAGGAGTTTC-30) that targets nucleo-tides 1233e1253 of HER-2 mRNA. We chose a 20-mer long oligo-nucleotide since this is the minimal length that would ensure specific binding to target mRNA. The specificity of the sequence was assessed by NCBI BLAST (basic local alignment search tool) analysis which revealed that the probe binding sequence is specific to homo sapiens HER-2 mRNA.