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NMR structural studies of intramolecular (Y+)_n•(R+)n(Y-)n DNA triplexes in solution: imino and amino proton and nitrogen markers of G.cntdot.AT base triple formation

Radhakrishnan, Ishwar and Gao, Xiaolian and de los Santos, Carlos and Live, David and Patel, Dinshaw J. (1991) NMR structural studies of intramolecular (Y+)_n•(R+)n(Y-)n DNA triplexes in solution: imino and amino proton and nitrogen markers of G.cntdot.AT base triple formation. Biochemistry, 30 (37). pp. 9022-9030. ISSN 0006-2960. doi:10.1021/bi00101a016.

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We reported previously on NMR studies of (Y+)_n•(R+)_n(Y-)_n DNA triple helices containing one oligopurine strand (R)_n and two oligopyrimidine strands (Y)_n stabilized by T-AT and C^+•GC base triples [de los Santos, C., Rosen, M., & Patel, D. J. (1989) Biochemistry 28, 7282-7289]. Recently, it has been established that guanosine can recognize a thymidineadenosine base pair to form a G•TA triple in an otherwise (Y+)_n•(R+)_n(Y-)_n triple-helix motif. [Griffin, L. C., & Dervan, P. B. (1989) Science 245, 967-971]. The present study extends the NMR research to the characterization of structural features of a 31-mer deoxyoligonucleotide that folds intramolecularly into a 7-mer (Y+)_n•(R+)_n(Y-)_n triplex with the strands linked through two T_5 loops and that contains a central G•TA triple flanked by T•AT triples. The G•TA triplex exhibits an unusually well resolved and narrow imino and amino exchangeable proton and nonexchangeable proton spectrum in H_2O solution, pH 4.85, at 5°C. We have assigned the imino protons of thymidine and amino protons of adenosine involved in Watson-Crick and Hoogsteen pairing in T•AT triples, as well as the guanosine imino and cytidine amino protons involved in Watsonxrick pairing and the protonated cytidine imino and amino protons involved in Hoogsteen pairing in C^+•GC triples in the NOESY spectrum of the G•TA triplex. The NMR data are consistent with the proposed pairing alignment for the G•TA triple where the guanosine in an anti orientation pairs through a single hydrogen bond from one of its 2-amino protons to the 4-carbonyl group of thymidine in the Watson-Crick TA pair. We detect a set of NOES between adjacent triples that establishes that the G•TA triple stacks between flanking T•AT triples in the G•TA triplex. The imino protons of the G•TA triplex in H_2O, pH 4.85, broaden sequentially between 32 and 42 °C with the thymidine imino protons in the central G•TA triple most stable to increasing temperature. The intramolecular G-TA triplex is stable at pH 5.0 and 5 °C but converts fully to the Watson-Crick hairpin duplex on raising the pH to 6.5 at this temperature. The assigned guanosine and thymidine imino protons have been linked to their attached ring nitrogens in the G•TA triplex from an analysis of the proton-detected natural abundance nitrogen-proton two-dimensional correlation spectrum. The guanosine N 1 nitrogen in the G•TA triple resonates 10 ppm to high field of the loop thymidine N3 nitrogens in contrast to their attached imino protons, which exhibit similar chemical shifts. These results demonstrate the capabilities of the NMR approach in monitoring individual base triples and their pairing alignments, as well as establishing that the G•TA triple can be readily accommodated in an otherwise intramolecular (Y+)_n•(R+)_n(Y-)_n triple helix in solution.

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Additional Information:© 1991 American Chemical Society. Published in print 17 September 1991. This research was supported by NIH Grant GM34504 to D.P. and NIH Grant DK38676 to D.L. C.d.1.S. was supported by NIH Fogarty Fellowship SF05 TWO41 69. The NMR spectrometers were purchased from funds donated by the Robert Woods Johnson Trust toward setting up an NMR center in the basic medical sciences at Columbia University. Purchase of a GN-500 spectrometer at Emory University was supported in part by NSF Grant DMB8604304 to D.L. We thank Karen Greene for technical assistance in the heteronuclear proton-nitrogen HMQC two-dimensional experiments.
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NIHSF05 TWO41 69
Robert Woods Johnson TrustUNSPECIFIED
Issue or Number:37
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Official Citation:NMR structural studies of intramolecular (Y+)n.cntdot.(R+)n(Y-)n DNA triplexes in solution: imino and amino proton and nitrogen markers of G.cntdot.AT base triple formation Ishwar Radhakrishnan, Xiaolian Gao, Carlos De los Santos, David Live, and Dinshaw J. Patel Biochemistry 1991 30 (37), 9022-9030 DOI: 10.1021/bi00101a016
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ID Code:84699
Deposited On:07 Feb 2018 16:25
Last Modified:15 Nov 2021 20:22

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