Sequence Specific Fluorescence Detection of Double Strand DNA

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Abstract
Methods for the fluorescent detection of specific sequences of double strand DNA in homogeneous solution may be useful in the field of human genetics. A series of hairpin polyamides with tetramethyl rhodamine (TMR) attached to an internal pyrrole ring were synthesized, and the fluorescence properties of the polyamide−fluorophore conjugates in the presence and absence of duplex DNA were examined. We observe weak TMR fluorescence in the absence of DNA. Addition of ≥1:1 match DNA affords a significant fluorescence increase over equimolar mismatch DNA for each polyamide−TMR conjugate. Polyamide−fluorophore conjugates offer a new class of sensors for the detection of specific DNA sequences without the need for denaturation. The polyamide-dye fluorescence-based method can be used to screen in parallel the interactions between aromatic ring pairs and the minor groove of DNA even when the binding site contains a non-Watson−Crick DNA base pair. A ranking of the specificity of three polyamide ring pairsPy/Py, Im/Py, and Im/Imwas established for all 16 possible base pairs of A, T, G, and C in the minor groove. We find that Im/Im is an energetically favorable ring pair for minor groove recognition of the T·G base pair.