DNA three-way junction

Three-Dimensional NOESY-NOESY Hybrid-Hybrid Matrix Refinement of a DNA Three-Way Junction... 

The methodology was tested on simulated data, derived from the Dickerson dodecamer, and used to refine the structure of a DNA three way junction from experimentally determined 3D NOESY-NOESY volume data. Our results indicate that the 3D hybrid-hybrid MORASS methodology, by combining the spectral dispersion of 3D NOESY-NOESY spectroscopy and the computational efficiency of 2D refinement programs, provides an accurate and robust means for structure determination of large biomolecules. 

In this paper, we report the hybrid-hybrid relaxation matrix refinement tested by simulated refinement calculations on a dodecamer DNA duplex, and the refinement of the three-dimensional structure of a DNA three-way junction (TWJ), using experimental 3D NOESY-NOESY data. The TWJ has previously been studied by 2D NMR spectroscopy (12, 13). 

Figure 3. The sequence of the DNA three-way junction molecule with two unpaired bases in the junction region.

Figure 7. The Final Structure of the DNA Three-Way Junction molecule. Only bonds between heavy atoms are shown.

Figure 11. A double-helical, DNA three-way junction (TWJ) with the Gibbs free energy of formation of the individual arms and the complete complex. Adapted from Reference [95].

Figure 13. Excess heat capacity (ACp) vs temperature curves for the denaturation of a DNA three-way junction and the deconvoluted enthalpy contributions of the three arms at (A) pH 5 and (B) pH 4.5 (1 M NaCl, 20 mM Na ro4). Adapted from Reference [33],...

Single-stranded DNA or RNA may adopt hairpin structures in which the distance between two sequences is much shorter than in the absence of hairpin. Figure B9.4.1 shows two synthetic targets, both containing 45 nucleotides, but only the first one is able to form a hairpin via a loop of four thymines. The second one is used as a control. Both contain the complementary sequences for ethidium-13-mer and 11-mer-coumarin separated by the same number of bases. The efficiency of energy transfer from coumarin to ethidium is dose to zero for the control, whereas it is about 25% in the hairpin structure. This value is low but the spatial conformation of this particular three-way junction is only partially known, and the transfer efhdency depends on the relative orientation and/or distance between coumarin and ethidium. 

Fig. 10 AFM analysis of three-way junction dynamics in liquid [48]. Reprinted with permission. Ten DNA molecules in the field are munbered. Molecules 7 and 9 (circled), in particular, change shape dramatically...

MB has been shown useful also for detection of cocaine by means of specific DNA aptamer [50]. The MB-tagged aptamer has been immobilized via thiol group onto a gold support. In absence of cocaine, the aptamer was partially unfolded. Addition of cocaine resulted in folding of aptamer into three-way junction, moving MB to a close proximity with the electrode surface. This resulted in an increase in reduction peak measured by AC voltammetry. Sensor was regenerable and allowed to detect cocaine within several seconds with sensitivity below 10pmol/L. 

Figure 7.5 Analysis of the two three-way RNA junctions of the VS ribozyme by comparative gel electrophoresis. The secondary structure of the VS ribozyme is shown, with the sequences of the two component three-way junctions. Each was analyzed in isolation by comparative gel electrophoresis, comparing the mobilities of the three long-short arm species. As before, these species have a central core of RNA that is extended with DNA sections. The junction species were electrophoresed in 10% polyacrylamide gels in the presence of 90 mM Tris—borate (pH 8.3) with 3 (junction III—IV—V) or 5 (junction II—III—VI) mM Mg2. The structural interpretations of both sets of data are shown. Both junctions undergo coaxial stacking of two arms, with die third directed laterally.

Fig. 2 Helicate which bind on three-way junction on DNA [37]. (a) Synthesis, (b) top and (c) side view of DNA and helicate...

In another example of molecular capsule synthesis, DNA-based capsule formation has been reported. In the example shown in Fig. 3.31, a three-way junction was formed from an equimolar mixture of three programmed oligo-DNA chains. Since this junction had attractive sticky ends , the mixture then further self-assembled into a nanocage. 

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