Parallel reaction pathways accelerate folding of a guanine quadruplex

Abstract

G-quadruplexes (G4s) are four-stranded, guanine-rich nucleic acid structures that can influence a variety of biological processes such as the transcription and translation of genes and DNA replication. In many cases, a single G4-forming nucleic acid sequence can adopt multiple different folded conformations that interconvert on biologically relevant timescales, entropically stabilizing the folded state. The coexistence of different folded conformations also suggests that there are multiple pathways leading from the unfolded to the folded state ensembles, potentially modulating the folding rate and biological activity. We have developed an experimental method for quantifying the contributions of individual pathways to the folding of conformationally heterogeneous G4s that is based on mutagenesis, thermal hysteresis kinetic experiments and global analysis, and validated our results using photocaged kinetic NMR experiments. We studied the regulatory Pu22 G4 from the c-myc oncogene promoter, which adopts at least four distinct folded isomers. We found that the presence of four parallel pathways leads to a 2.5-fold acceleration in folding; that is, the effective folding rate from the unfolded to folded ensembles is 2.5 times as large as the rate constant for the fastest individual pathway. Since many G4 sequences can adopt many more than four isomers, folding accelerations of more than an order of magnitude are possible via this mechanism.

Folding dynamics of polymorphic G‐quadruplex structures

“…Mittermaier et al have presented a sophisticated strategy to extract dynamic information from experiments under deliberately chosen conditions that provoke thermal hysteresis. [30,69,73,76] Rapid temperature jumps (T-jump) have been used to induce G4 folding in circular dichroism (CD)-spectroscopic [77] and mass spectrometric [78] setups and new probe designs could potentially allow T-jump induced folding also for NMR spectroscopy. [79][80][81] Thermal (un-)folding is typically limited to lower than physiological K + concentrations, due to the high thermal stability of many G4 structures.…”

“…This concept was first exploited to study RNA refolding, [89][90][91][92] and has been applied to G4s recently in two different ways, either by selecting single folded conformations out of a polymorphic conformational ensemble from a G4 forming sequence instead of using sequence mutations (Figure 2E) [69] ; or to suppress completely the G4 folding to trap the unfolded state (Figure 2D). [33,76] The method allows investigating the isothermal folding at constant experimental conditions and ensures a robust disruption of hydrogen bond interactions or preferential pre-orientations. While photocages are introduced at the nucleobases and act irreversible in the direction of folding, the incorporation of photosensitive scaffolds into the DNA backbone can be used to cleave and hence unfold G4s (Figure 2F), [93] or enable a reversible switching between different G4 conformational states.…”

“…The possibility to fold into the direction of a fourfold basin with an increased conformational space thus compensates for the entropy penalty during folding resulting in an accelerated concurrent overall folding for 2345. [76] We denote the G-register isomers of 2345 with respect to the G-tracts 3 (first digit) and 5 (second digit) in a two digit abbreviation depending if the registers are shifted in 5 0or 3 0 -direction, resulting in conformations 33, 35, 55 and 53. [30,69,76] Out of the four possible G-register isomers, only two are majorly populated in the wildtype sequence at thermal equilibrium, namely conformations 33 and 53.…”

Transient equilibrium mapping: A new tool for measuring the thermodynamics of slowly assembling supramolecular systems

“…Common examples include amyloid fibrils, viral capsids, and a variety of self-assembling non-biological small molecules. [10][11][12][13][14][15][16] Thermodynamic analyses rely on assembled and disassembled monomers existing at their equilibrium distributions over the range of temperature measured during the experiment. Slowly assembling systems require so much time to equilibrate that this is not achievable on practical timeframes using standard melting and annealing experiments.…”

Time-resolved NMR-spectroscopic studies of conformational dynamics in DNA G-quadruplexes

“…This chapter relates to the major findings of the publication: "Parallel reaction pathways accelerate folding of a guanine quadruplex" (Harkness, Hennecker, Grün et al NAR 2021). 752 This publication contains contributions from me (the author of this thesis) using the photocaging approach for lightinduced folding.…”