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Jon Pokorski, NIDDK
Friday,
Dec.
2, 2005 (S,S)-trans-Cyclopentane PNA: An Optimized Probe for Nucleic Acid Detection
Abstract: Peptide nucleic acids are nucleic acid analogues in which the sugar-phosphate backbone is replaced with an uncharged, achiral backbone. This backbone is derived from ethylene diamine and as such is termed aminoethyl glycine PNA (aegPNA). Peter Nielsen and coworkers introduced this backbone in an effort to develop a major groove binder that would form a triple helix with double stranded DNA1. The aegPNA, however, proved to be a much more general molecule, binding to both DNA and RNA with significantly higher affinity than the natural counterparts. In unmodified PNAs, the aminoethylglycine backbone is flexible and can adopt many conformations. As a result, there is a significant entropic loss associated with duplex formation. Restricting rotation about the C2-C3 bond should decrease this loss and further favor hybridization. With this in mind, trans-1, 2-cyclopentane diamine and trans-1, 2-cyclopropane diamine have been introduced into the PNA backbone. The cyclopropane modification exhibited equivalent stability to the aegPNA of corresponding sequence, and hence was not optimal for further pursuit. Alternatively, the cyclopentane modification increases oligonucleotide binding affinity and mismatch discrimination compared to unmodified PNAs. These improvements are shown to be independent of nucleobase composition and sequence, as well as being highly dependent on the (S, S) stereochemistry. These properties make cyclopentane PNA (tcypPNA) optimal for hybridization-based applications. tcypPNAs have been immobilized onto a glass surface and implemented in an established nano-particle based DNA detection assay. The sensitivity of the assay has been improved by three orders of magnitude over published results, which used DNA as the capture probe, detecting synthetic anthrax lethal factor DNA at a 50 aM level (aM = 10-18 M). tcypPNA was also shown to maintain a high level of specificity in target binding. This translated to a selectivity of three to one for the target DNA relative to the non-complementary single base mismatch sequence.
Jon received his B.S. degree in biochemistry from UCLA. In 2002, he joined the department of chemistry at Northwestern University as a graduate student. In 2005, he moved with the Appella group to the NIH where he is continuing to work on his research toward his Ph.D. |
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