Applying the appropriate model and nonlinear regression in
data analysis, ITC can determine the association constant or binding affinity, K a , the binding enthalpy, ΔH, the stoichiometry, n, the
entropy change, ΔS, and the Gibbs free energy of binding, ΔG, in a
single experiment. In contrast, spectroscopy experiments must be
performed at various temperatures to determine the enthalpy and
entropy of the reaction. It should be noted that the heat measured
upon RNA-ligand binding does not result only from the formation
of the direct RNA-ligand interactions but also includes heat generated from other binding-associated events, such as allosteric structural transitions and desolvation of the molecules. While several
methodological advances allowed ITC to be used for determining
cooperativity in protein systems [18, 19], the use of calorimetry to
directly determine cooperativity of ligand binding to RNA is difficult [20], which makes ITC more appropriate for studying the
mechanisms of RNA folding [21] and macromolecular assemblies
[22, 23].
Spectroscopic and ITC methods typically provide bulk measurements and are invaluable for determining macroscopic characteristics of RNA systems but cannot directly visualize
conformational transitions in RNA folding. The allosteric modulations can, however, be traced at the level of individual molecules by
the so-called single-molecule techniques such as single-molecule
fluorescence resonance energy transfer (smFRET) [24]. This
method involves visualization of fluorescently labeled individual
RNA molecules under the microscope. Special fluorescent labels
attached to different regions of RNA induce FRET when coming in
close proximity upon ligand binding [25]. Analysis of individual
traces of molecules provides a comprehensive picture of allosteric
re-arrangements in the RNA.
2.3 Determination
of Cooperative Ligand
Binding “On the Fly”
Throughout the twentieth century, researchers have developed
various models to describe the binding of multiple ligands to
oligomeric proteins, and many such models are applicable to manifestations of cooperativity in RNA. One of the most used models
describing cooperative binding of ligands was developed by Hill
and named after him [26]. The equation produces a “Hill coefficient” n, which is more than 1 when the system exhibits positive
cooperativity and less than 1 if the system exhibits negative cooperativity, with the total number of ligand-binding sites being an
upper limit for the coefficient. Although not ideal for evaluation of
cooperativity [27, 28], the Hill coefficient is broadly used by biochemists for detecting cooperativity in the systems that involve
binding of multiple ligands to RNA molecules. For example, the
Hill coefficient value of 1.6 was the basis for conclusions about
positive cooperativity in the truncated version of the dual glycinesensing RNA [29].
260
Alla Peselis and Alexander Serganov
data analysis, ITC can determine the association constant or binding affinity, K a , the binding enthalpy, ΔH, the stoichiometry, n, the
entropy change, ΔS, and the Gibbs free energy of binding, ΔG, in a
single experiment. In contrast, spectroscopy experiments must be
performed at various temperatures to determine the enthalpy and
entropy of the reaction. It should be noted that the heat measured
upon RNA-ligand binding does not result only from the formation
of the direct RNA-ligand interactions but also includes heat generated from other binding-associated events, such as allosteric structural transitions and desolvation of the molecules. While several
methodological advances allowed ITC to be used for determining
cooperativity in protein systems [18, 19], the use of calorimetry to
directly determine cooperativity of ligand binding to RNA is difficult [20], which makes ITC more appropriate for studying the
mechanisms of RNA folding [21] and macromolecular assemblies
[22, 23].
Spectroscopic and ITC methods typically provide bulk measurements and are invaluable for determining macroscopic characteristics of RNA systems but cannot directly visualize
conformational transitions in RNA folding. The allosteric modulations can, however, be traced at the level of individual molecules by
the so-called single-molecule techniques such as single-molecule
fluorescence resonance energy transfer (smFRET) [24]. This
method involves visualization of fluorescently labeled individual
RNA molecules under the microscope. Special fluorescent labels
attached to different regions of RNA induce FRET when coming in
close proximity upon ligand binding [25]. Analysis of individual
traces of molecules provides a comprehensive picture of allosteric
re-arrangements in the RNA.
2.3 Determination
of Cooperative Ligand
Binding “On the Fly”
Throughout the twentieth century, researchers have developed
various models to describe the binding of multiple ligands to
oligomeric proteins, and many such models are applicable to manifestations of cooperativity in RNA. One of the most used models
describing cooperative binding of ligands was developed by Hill
and named after him [26]. The equation produces a “Hill coefficient” n, which is more than 1 when the system exhibits positive
cooperativity and less than 1 if the system exhibits negative cooperativity, with the total number of ligand-binding sites being an
upper limit for the coefficient. Although not ideal for evaluation of
cooperativity [27, 28], the Hill coefficient is broadly used by biochemists for detecting cooperativity in the systems that involve
binding of multiple ligands to RNA molecules. For example, the
Hill coefficient value of 1.6 was the basis for conclusions about
positive cooperativity in the truncated version of the dual glycinesensing RNA [29].
260
Alla Peselis and Alexander Serganov