Probing Sequence Specificity

Analysis of the pyrene-labeled homoduplex 5 5 (Eig. 9.3b) by NMR, mass spectrometry, and TLC suggested that 5 5 had a stability similar to that of 3 4. NOESY spectra revealed cross-strand NOEs consistent with the formation of the self-dimer 5 5 (Zeng et al. 2003). Based on a fluorescence method described in the literature (Sontjens et al. 2000), the dimerization constant of the pyrene-labeled duplex 5 5 was found to be (6.77 + 4.12) x 10 M. The studies on duplexes 3 4 and 5 5 clearly demonstrated that the stabilities of our duplexes are indeed only determined by the number of intermolecular H bonds, and both hetero- and homoduplexes can be easily designed and constructed. 

the stability is enhanced by more than 5 orders of magnitude with two additional H bonds (i.e., the quadmply vs. 6-H bonded duplexes), which implies the presence of positive cooperativity in this H bonded system. Comparing the stabilities of doubly (Aia = 25 -0.9 kcal/mol for each H bond) and quadmply (dimers of 

Sequence-specific pairing of DNA and RNA strands is essential for the storage, transmission, and expression of genetic information, which forms the basis for techniques such as polymerase chain reaction, hybridization techniques, and DNA chip arrays. Having demonstrated the sequence-independent namre in the stabilities of 

The NOESY spectrum of 3 6 showed that one end of this pair was locked by intermolecular H bonds, whereas the other end around the mismatched site consisting of the two amide carbonyl groups at that end was open. In contrast, the NOESY study indicated that both ends of 3 7 were locked, but no or very weak NOEs were detected for protons close to the internal mismatched site. These results indicated that, in spite of the presence of mismatched sites, these mutant duplexes still formed. More detailed analysis showed five interstrand H bonds in 3 6 or 3 7, suggesting that these strands were still fully registered. However, the oli-goamide backbones must be twisted near the mismatched sites to alleviate any unfavorable interactions. 

The sequence specificity in the formation of duplex 3 4 was probed in the presence of strands 6 and 7 by H-NMR. Thus, adding 1 equiv of 4 into the solution of either 3 6 or 3 7 in CDCI3 containing 5% dimethylsulfoxide-iig (DMSO-iie) led to the complete replacement of the wrong strands 6 or 7, resulting in the appearance of new H-NMR signals corresponding to those from duplex 3 4 and the free strand 6 or 7. 

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Nakayama, S. Yan, L. Sintim, H. O. Junction probes - sequence specific detection of nucleic acids via template enhanced hybridization processes. J. Am. Chem. Soc. 2008,130,12560-12561. 

Saiki RK, Walsh PS, Levbnson CH., Erlich HA (1989) Genetic analysis of amplified DNA with immobilized sequence specific oligonucleotide probes. Proc. Natl Acad Sd USA 86 6230-6234. 

Capture molecule = probe] Gone-specific sequence,... 

Bugawan, T.L., Begovich, A.B., and Erlich, H.A. (1990) Rapid HLA-DPB typing using enzymatically amplified DNA and nonradioactive sequence-specific oligonucleotide probes. Immunogenetics 32, 231-241. 

Haugland, R. A., and Heckman, J. L. (1998). Identification of putative sequence specific PCR primers for detection of the toxigenic fungal species Stachybotrys chartarum. Mol. Cell. Probes 12, 387- 96. 

Holloway, J. W. Beghe, B. Turner, S. Hinks, L. J. Day, I. N. Howell, W. M. Comparison of three methods for single nucleotide polymorphism typing for DNA bank studies sequence-specific oligonucleotide probe hybridisation, TaqMan liquid phase hybridisation, and microplate array diagonal gel electrophoresis (MADGE). Hum. Mutat. 1999,14(4), 340-347. 

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