SUNY at Albany
June 19-23, 2001
Thermal Stability of the Parallel Triplex Motif: Influence of Cytosine Modifications
Triplex DNA can be formed by the binding of a third nucleic acid strand in the major groove of a duplex DNA. In the so-called classical parallel motif (YRY) the third, homopyrimidine (Y) strand has parallel orientation to the homopurine (R) strand of the duplex forming triads TAT and C+GC. The positive charge of the cytosines in the third strand is necessary to enable Hoogsteen base pairing with guanine. However, YRY-triplex stability at neutral pH can be realized by substitution of cytosines of the single strand to 5?-methylcytosines (mC) or to 2?-O-methyl-pseudoisocytosines (pC).
We have found a new parallel triplex motif stable at neutral pH. Hairpin duplexes are synthesized as 12-nt complementary strands with Watson-Crick base pairing linked by four cytosines. In the triplex a single third strand is identic to the purine-rich duplex strand and these both homologous strands have parallel orientation. The strands consist of mixed purine-pyrimidine sequences unlike the monotonous homopurine and homopyrimidine motifs usual for triple-stranded DNA.
In the present study the stability of various DNA hairpin duplexes with corresponding single strands in mixtures 1:1 of the components is investigated by UV-melting. Triplex formation is supported by the mixing curves, UV-hypochromicity data and CD-spectra. Cary-100 spectrometer is used for UV-measurements. Melting temperatures (Tm) are determined from the first derivatives of melting profiles. Melting profiles (absorbance vs. temperature) are obtained at 260 nm from 11° C to 91° C at 0.2K increment with heating rate of 1.0° /min. All the results are related to 2.0 mM strand concentration. The solvent consists of as: 200mM NaCl, 10mM sodium cacodylate, 0.1mM EDTA, pH 7.3.
UV-melting curves are biphasic. Tm1 values of triplexes (the first transition in the UV-melting curve) are for 22 K ? 27 K lower, than Tm2 ones of duplex hairpin. The values of Tm2 for triplexes are equal to Tm for corresponding hairpin duplexes in absence of the single strands within the precision of the measurements. The stability of the triplexes (Tm1) depends on the percent of pyrimidines in the purine-rich strand and on the sequence.
The replacement of several cytosines of the single strand by mC stabilizes the complexes. These data are in agreement with the stabilization of C+GC triads in the YRY, when mC replaces C+ in the third strand. The substitution of cytosines in the single strands by pC has expressed destabilizing effect in contrast to the YRY. Obviously pC-modification is unfavorable for the complex formation of this type. These results are discussed proceeding from the model for this parallel triplex motif. We assume that the structure of the base triad CGC corresponds to that in recombination motif of Zhurkin and co-authors.
Addition of 10 µM benzo[e]pyridoindole derivative (BePI), which is known as a specific stabilizing ligand for triplexes, increases also stability of the studied complexes as in unmodified as well as in modified forms. For the investigated systems the Tm1 values increase also after addition of magnesium ions (10 mM) as an unspecific triplex stabilizer. There is a distinct sequence dependence of all the observed effects.
This study was supported by the DFG, DAAD and Funds of the Chemical Industry (Germany).
Anna Gabrielian(1)*, Axel Walter(2), Holger Schütz(2), Eckhard Birch-Hirschfeld(3), R.Dayal Yadav(4), Shwu-Bin Lin(5), Wei-Chen Lin(4), Lou-Sing Kan(4) and Hartmut Fritzsche(2)*
Institue of Molecular Biology,