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 pairs-Py/Py, Im/Py, and Im/Im-was 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.
The host factor LSF represses the human immunodeficiency virus type 1 long terminal repeat (LTR) by mediating recruitment of histone deacetylase. We show that pyrrole-imidazole polyamides targeted to the LTR can specifically block LSF binding both in vitro and within cells via direct access to chromatin, resulting in increased LTR expression.
We identify histone deacetylation as a mechanism that can dampen viral expression in infected, activated CD4+ T cells and establish a persistent, quiescent reservoir of HIV infection.
Reactive ion etching (RIE) was used to pattern antibodies onto the surfaces of polymer substrates. A low pressure, inductively coupled oxygen plasma was used to anisotropically etch 25-30 mum deep features into poly(methyl methacrylate) (PMMA), Zeonex, and polycarbonate (PC). Scanning electron microscopy and contact angle measurements show that the resulting surfaces exhibit significant microroughness and enhanced hydrophilicity. Fourier transform infrared spectroscopy suggests that, in addition to enhanced surface area, chemical modifications may contribute to antibody immobilization. Polyclonal antibodies preferentially bind to the etched areas in RIE-patterned PMMA and Zeonex substrates but localize in unetched regions of RIE-patterned PC surfaces. Simple immunoassays were performed to demonstrate a potential application for RIE-modified polymer surfaces. Antibodies specific for the capture of fluorescently labeled cholera toxin, S. aureus enterotoxin B, and B. anthracis protective antigen were immobilized onto etched PMMA surfaces and shown to specifically capture their labeled antigen from solution. This work demonstrates a potentially useful fabrication methodology for constructing antibody microarrays on plastic substrates.
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