We use the Atto dyes Atto 550 and Atto 647N.
DAPI is an unfused aromatic ring system linked by bonds with torsional freedom which allow the molecules to adopt appropriate conformation to fit the helical curvature of the groove without significant perturbation of the DNA. DAPI has demonstrated DNA binding via intercalation with poly[d(GC)]. It is a nonclasical intercalator. (Strekowski et.al.)
Lots of small molecules bind to DNA, with highly diverse effects. Nevertheless, most compounds can be classified according to their binding mode: major groove binder, minor groove binder, or intercalator. The last ones insert between neighbored bases. Here we examined one example for each of these groups:
|DAPI||minor groove binding||0.9 nM
|Spermine||major groove binding||4.8 µM
|Ethidium bromide||intercalation||12.0 µM
Ethidium bromide is a classical intercalator. It is a fused-ring aromatic molecule with positive charges on an attached side chain and/or the ring system itself. The planar ring system stacks well with base pairs in the intercalation. Ethidium bromide intercalates from the minor groove of the duplex complex. (Strekowski et.al.)
FOB is short for folding buffer. It contains 50mM Tris and 10mM EDTA. FOBxM refers to FOB without MgCl2
, whereas FOB20 is FOB with 20mM MgCl2
In short, FRET is a fluorescence based approach often used in biosciences to measure distances in the lengthscale of up to ca. 10nm. For more explanation, please read the entry in the methods section.
Intercalation is known to occur without interfering with hydrogen bonding of the base pairs and obeys the nearest neighbor exclusion principle. When DNA is saturated with intercalators, every second potential intercalation site on the helix remains empty. In order for intercalation to ensue, theDNAbase pairs separate 3.4 °A to form a cavity for the incoming planar ligand through localized left-handed unwinding of the duplex. The normal twist of B-formDNA is 36◦ (10 base pairs per one turn of 360◦). In order to accommodate the ligand, a reduction of this rotation occurs. The unwinding angle varies with the geometry of the ligand–DNA complex.
In spite of the wealth of structural data known for intercalation complexes, the origin of the intercalation forces is not fully understood. Intercalation has been generally considered to be the result of a hydrophobic interaction in which a hydrophobic aromatic molecule is drawn to a hydrophobic environment of the base pairs from the hydrophilic aqueous environment.
Spermine is a polyamine, which is protonated under physiological conditions and therefore interacts strongly with DNA. All theoretical and experimental results (see: Noncovalent interactions with DNA: An overview) can be rationalized in terms of the concept of the negative potentials in the DNA macromolecule. It has been concluded that the highest concentration of the negative potentials is in the grooves of the DNA duplexes rather than on the backbones, despite the location of the anionic phosphates within these backbones. The negative potentials are stronger in the major groove than in the minor groove at GC sequences. Accordingly, a flexible cationic molecule of protonatedspermine forms a complex within the DNA major
groove with GC sequences following the negative potential concentration. (Strekowski et.al.)
Transmission Electron Microscopy (TEM)
Transmission electron microscopy is an imaging technique to observe thin specimen. An electron beam interacts with the specimen while passing and is detected by a sensor.
Organic samples can be prepared with high atomic number stains to enhance contrast. Electrons can pass through the biological structure and are absorbed or scattered by the stain.