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Albany 2001

category image Biomolecular
Stereodynamics
SUNY at Albany
June 19-23, 2001

Formation of Intramolecular DNA Triplex Monitored by Fluorescence of 2-aminopurine Incorporated in the Third Strand

Three-stranded nucleic acids alignments occur in the course of various biological processes, such as recombination. The parallel (recombination) "R-triplex" was predicted theoretically (1) and studied experimentally using the protein-free intramolecular (2) and intermolecular (3) oligonucleotide models. Although the detailed 3D structure of this hypothetical "triplex" has been visualized only for the G*(C?G) and C*(G?C) triplets so far (4), in principle such a fold can accommodate any nucleotide sequence (with the two identical DNA strands, W and R, in parallel orientation). Here, we show that the fluorescent base 2-aminopurine (2AP) can be used as a structural probe for monitoring formation of the AT-containing triplex.

Deoxyribooligonucleotides potentially able to form the intramolecular R-triplex (RCW and R-2AP) or the duplex (CW and ds-2AP) were studied: RCW: 5'-GTAGACTGAG TTTT CTCAGTCTACGC GAA GCGTAGACTGAG-3'
R-2AP: 5'-GTaGACTGAG TTTT CTCAGTCTACGC GAA GCGTAGACTGAG-3'
CW: 5'-CTCAGTCTACGC GAA GCGTAGACTGAG-3'
ds-2AP: 5'-GTaGACTGAG TTTT CTCAGTCTAC-3'
ss-2AP: 5'-GTaGACTGAG-3'
(where a stands for 2AP; the single strand ss-2AP was used as a control). The GAA linker together with the two adjacent GC pairs would stabilize the duplex part of RCW and R-2AP, whereas the flexible TTTT loop connects the third strand to the duplex. 2AP is known to form a base pair with thymine, and potentially can substitute for adenine in the A*(T?A) triplet, forming 2AP*(T?A) triplet consistent with the R-form (1). Formation of the intramolecular triplexes RCW and R-2AP was observed using two methods: (i) UV absorption as a function of temperature, and (ii) fluorescence polarization of the intercalated EtBr probe. Here we demonstrate that the triplex fold can be independently detected by the fluorescence of 2AP.

Fluorescence emission of the 2AP at 370 nm (excitation wavelength 310 nm) was found to be distinctly different for the three studied oligonucleotides (see Figure). At 3ºC (0.5 M LiCl, tris pH 7.6), the relative quantum yield of 2AP fluorescence (RQY) in the folded R-2AP triplex was 1.6 times higher than in the ds-2AP duplex, and 1.7 times less than in the single strand ss-2AP. The smooth decrease in the ss-2AP fluorescence in the temperature range 3-43ºC is likely due to the temperature dependence of the quantum yield of the unpaired 2AP. In this range, the ds-2AP remains in the double-stranded conformation, with the premelting starting above 35-38ºC (as revealed with UV temperature profiles; data not shown). Accordingly, the changes of fluorescence of 2AP incorporated in the duplex are negligible at 3-38ºC. The sigmoidal temperature dependence of RQY in the case of R-2AP apparently reflects a conformational change in the structure. Importantly, this conformational transition occurs in the same temperature range as the unstacking of the bases in the third strand, detected independently by UV hyperchromicity. Therefore, we attribute the transition in R-2AP to the melting of the three-stranded structure to a structure with a "dangling" third strand and unstacked 2AP bases. Upon completion of the "melting" above 30-32ºC, the RQY value for the R-2AP becomes close to that for the single strand ss-2AP. Note that the duplex part (CW) of the intramolecular triplex remains intact in this temperature interval.

The fluorescence of 2AP nucleotide is well known to quench considerably upon shielding from solvent due to the stacking and hydrogen-bonding interactions with other bases (see the ss-2AP and ds-2AP temperature profiles in the Figure). Thus, by comparing the relative quenching of the 2AP fluorescence in the R-2AP and in the ds-2AP (at low temperatures) we suggest that the 2AP fluorophore is more exposed to solvent in the R-2AP "triplex" compared to the ds-2AP duplex. The observed "melting" of a single 2AP*(TáA) triplet demonstrates the potential of 2AP as a structural probe for monitoring the rearrangement of nucleic acid strands upon formation and dissociation of three-stranded complexes.

References and Footnotes
  1. V.B. Zhurkin, G. Raghunathan, N.B. Ulyanov, R.D. Camerini-Otero, and R.L. Jernigan, J. Mol. Biol., 239, 181-200, 1994.
  2. A.K. Shchyolkina, E.N. Timofeev, O.F. Borisova, I.A. Il'icheva, E.E. Minyat, E.E. Khomyakova, V.L. and Florent'ev, FEBS Letters, 339, 113-118, 1994.
  3. A.K. Shchyolkina, E.N. Timofeev, Yu.P. Lysov, V.L. Florentiev, T.M. Jovin, and D.J. Arndt-Jovin, NAR, 29, 986-995, 2001.
  4. B.T. Wimberly, R. Guymon, J.P. McCutcheon, S.W. White and V. Ramakrishnan, Cell, 97, 491-502, 1999.

The study was partially supported by the RFBR grants N 99-04-49179 and N 01-04-48561.

Dmitry N. Kaluzhny (1), Olga F. Borisova (1), Robert L. Jernigan (2), Victor B. Zhurkin (2), Anna K. Shchyolkina (1)*

Engelhardt Institute of Molecular Biology RASc(1), Vavilova 119991 Moscow,
Russia and National Cancer Institute(2), NIH, Bethesda, MD, USA
* Tel: 7095-1359789; Fax: 7095-1351405; E-mail: annas@genome.eimb.relarn.ru