Mapping the energy landscapes of supramolecular assembly by thermal hysteresis

Abstract

Understanding how biological macromolecules assemble into higher-order structures is critical to explaining their function in living organisms and engineered biomaterials. Transient, partly-structured intermediates are essential in many assembly processes and pathway selection, but are challenging to characterize. Here we present a simple thermal hysteresis method based on rapid, non-equilibrium melting and annealing measurements that maps the rate of supramolecular assembly as a function of temperature and concentration. A straightforward analysis of these surfaces provides detailed information on the natures of assembly pathways, offering temperature resolution beyond that accessible with conventional techniques. Validating the approach using a tetrameric guanine quadruplex, we obtain strikingly good agreement with previous kinetics measurements and reveal temperature-dependent changes to the assembly pathway. In an application to the recently discovered co-assembly of polydeoxyadenosine (poly(A)) and cyanuric acid, we show that fiber elongation is initiated when an unstable complex containing three poly(A) monomers acquires a fourth strand.

The Unexpected Base‐Pairing Behavior of Cyanuric Acid in RNA and Ribose versus Cyanuric Acid Induced Helicene Assembly of Nucleic Acids: Implications for the Pre‐RNA Paradigm

“…Many of the biophysical datap resented in the previous work of the group of Sleiman [4,17] seem to be consistent with the helicene dyad model for the CA-mediated supramolecular assemblies of poly(A). The height of the fibers, as observed by the AFM data, [4] would also be consistentw ith ah elicene model.…”

“…[4,17] Furthermore, the robust elongation into micron-long polymers [4,17] fits wellw ith the helicene model, which naturallyr esultsi no verhangs (stickye nds) that would lead to as taggered assembly [4] and overlap between strands, creating the distribution of fiber lengths. [17] The opposing helicity of the backbone (righthanded) versus the helicene of the nucleobases (left-handed) may also explain the dramatic CD characteristic of CA-poly(A) systems( strongn egative bands at 212 and 252 nm, with weakly positive bands at 267 and 285 nm), as noted by Sleiman and co-workers. [4] Our helicene model suggests that as ig-nificant structuralc hange has taken place and should not be interpreted as implying anything more (especially with the overall handedness of the system).…”

Noncovalent Helicene Structure between Nucleic Acids and Cyanuric Acid

“…Experiments by Sleiman andc o-workerso nt he association between substituted adenosine monomersa nd substituted CA in toluene suggest at riplex formation. [29] However, melting temperature analysiso fv arious pre-organized constructs [29] and thermalh ysteresis experiments [47] are less conclusive in establishing triplex formation for the poly(A)-CA assemblies. Therefore, we believe that both the helicene-dyad and the helicene-triad model are consistentw ith the bulk of availablee xperimental dataa nd that they are both more likely than the hexad model for the DNA/RNA assemblies with CA.…”

Parallel reaction pathways accelerate folding of a guanine quadruplex

“…This G4 controls 80–90% of the expression of the potent c-myc oncogene ( 48 ), with c-myc estimated to be deregulated in >50% of human cancers ( 49 ). Our approach combines mutagenesis, thermal hysteresis (TH) kinetics melting experiments ( 50 , 51 ) and a novel global fitting procedure to dissect the kinetic contributions of individual pathways to the overall folding rate. We find that folding of the Pu22 G4 is accelerated by roughly a factor of 2.5 due to the existence of four parallel pathways, with a macroscopic folding rate about 2.5 times as large as that of the most rapid individual route.…”