Synthesis and Fluorescence Studies of Multiple Labeled Oligonucleotides Containing Dansyl Fluorophore Covalently Attached at 2-Terminus of Cytidine via Carbamate Linkage

Synthesis of modified oligonucleotides in which the specific cytidine nucleoside analogues linked at 2ʹOH position via a carbamate bond with an amino ethyl derivative of dansyl fluorophore is reported. For the multiple labeling of oligonucleotides, a strategy involving prelabeling at the monomeric level followed by solid phase assembly of oligonucleotides to obtain regiospecifically labeled probes has been described. The labeled monomer was phosphitylated using 2-cyanoethyl- N,N,Nʹ,Nʹ-tetraisopropyl-phosphoramidite (Bis-reagent) and pyridiniumtrifluoro acetate (Py·TFA) as an activator. To ascertain the minimal number of labeled monomers required for a specific length of oligonucleotide for detection and also to assess the effect of carbamate linkage on hybridization, hexamer and 20-mer sequences were selected. Both were labeled with 1, 2, and 3 monomers at the 5ʹ-end and hybridized with normal (unmodified) complementary sequences. As compared to midsequence or 3ʹ-terminal labeling reported earlier, the 5ʹ-terminal labeling has been found to have minimal contact-mediated quenching on duplex formation. This may be due to complementary deoxyguanosine (dG) rich oligonucleotide sequences or CG base pairs at a terminus that is known to yield stronger binding. This is one reason for selecting cytidine for labeling. The results may aid rational design of multiple fluorescent DNA probes for nonradioactive detection of nucleic acids.