Analysis of branched nucleic acid structure using comparative gel electrophoresis

Abstract

Electrophoresis in polyacrylamide gels provides a simple yet powerful means of analyzing the relative disposition of helical arms in branched nucleic acids. The electrophoretic mobility of DNA or RNA with a central discontinuity is determined by the angle subtended between the arms radiating from the branchpoint. In a multi-helical branchpoint, comparative gel electrophoresis can provide a relative measure of all the inter-helical angles and thus the shape and symmetry of the molecule. Using the long-short arm approach, the electrophoretic mobility of all the species with two helical arms that are longer than all others is compared. This can be done as a function of conditions, allowing the analysis of ion-dependent folding of branched DNA and RNA species. Notable successes for the technique include the four-way (Holliday) junction in DNA and helical junctions in functionally significant RNA species such as ribozymes. Many of these structures have subsequently been proved correct by crystallography or other methods, up to 10 years later in the case of the Holliday junction. Just as important, the technique has not failed to date. Comparative gel electrophoresis can provide a window on both fast and slow conformational equilibria such as conformer exchange in four-way DNA junctions. But perhaps the biggest test of the approach has been to deduce the structures of complexes of four-way DNA junctions with proteins. Two recent crystallographic structures show that the global structures were correctly deduced by electrophoresis, proving the worth of the method even in these rather complex systems. Comparative gel electrophoresis is a robust method for the analysis of branched nucleic acids and their complexes.

Structure of the three-way helical junction of the hepatitis C virus IRES element

“…Comparative gel electrophoresis provides a simple yet powerful way to examine the global conformation of a helical junction in RNA under a variety of conditions (Lilley 2008(Lilley , 2009a. The IRES junction was constructed from three separate strands (each generated by transcription from a DNA template), and prepared in different forms comprising the three possible variants with two long (40 base pairs [bp]) arms and one shortened (10 bp) arm.…”

Telomerase Activity Is Sensitive to Subtle Perturbations of the TLC1 Pseudoknot 3′ Stem and Tertiary Structure

“…It is interesting to note that both the 809 and 811 sites involve the 2′-Omethyl of pyrimidine (Py) nucleotides, while the 805 site involves a purine (Pu), since dPy nucleotides in the context of an RNA helix influence the intensity of the 210-nm band. 42,43 Consistent with their larger effects on the 210-nm band, the intensity decrease in the major positive band at 264 nm is larger for both the 809 and 811 backbone substitutions, although all three substitutions have a decrease in signal intensity at 264 nm, compared to the pure RNA duplex.…”

“…One possibility is that the alternative conformation reflects a different orientation of the stems at the helical junction or more likely the stem 2 helices above and below the loop that was trapped and separated in the gel. Different orientations of the helices with respect to each other would change the shape of the molecule and run differently on the gel, 43 without necessarily making large changes in the hydrogen bonding and stacking interactions that make up the bulk of the thermodynamic stability. Therefore, since we have no evidence for a contribution from the additional conformation to the melting profiles for these complexes, we have interpreted the data without including the minor conformer.…”

Hexapeptides That Inhibit Processing of Branched DNA Structures Induce a Dynamic Ensemble of Holliday Junction Conformations

“…Revealed by Gel Mobility Shifts-The square planar and stacked X conformations give characteristic electrophoretic gel mobilities, because the lengths of the DNA arms in conjunction with the angles subtended between adjacent arms determine the migration velocities (49). To complement the smFRET analysis of junction conformations and to relate the current work to previous analyses, we performed gel mobility shift analyses on the peptide-free and peptide-bound junctions.…”