Biology subjects

Abou Assi, H.

Publications and source records attributed to Abou Assi, H..

4 recordsLinked to original sources

bioRxiv2020

delta-Melt: Nucleic acid conformational penalties from melting experiments

Thermodynamic preferences to form non-native conformations are crucial for understanding how nucleic acids fold and function. However, they are difficult to measure experimentally because this requires accurately determining the population of minor low-abundance (<10%) conformations in a sea of other conformations. Here we show that melting experiments enable facile measurements of thermodynamic preferences to adopt non-native conformations in DNA and RNA. The key to this delta-melt approach is to use chemical modifications to render specific minor non-native conformations the major state. The validity and robustness of delta-melt is established for four different non-native conformations under various physiological conditions and sequence contexts through independent measurements of thermodynamic preferences using NMR. delta-melt is fast, simple, cost-effective, and enables thermodynamic preferences to be measured for exceptionally low-populated conformations. Using delta-melt, we obtained rare insights into conformational cooperativity, obtaining evidence for significant cooperativity (1.0-2.5 kcal/mol) when simultaneously forming two adjacent Hoogsteen base pairs. We also measured the thermodynamic preferences to form G-C+ and A-T Hoogsteen and A-T base open states for nearly all sixteen trinucleotide sequence contexts and found distinct sequence-specific variations on the order of 2-3 kcal/mol. This rich landscape of sequence-specific non-native minor conformations in the DNA double helix may help shape the sequence-specificity of DNA biochemistry. Thus, melting experiments can now be used to access thermodynamic information regarding regions of the free energy landscape of biomolecules beyond the native folded and unfolded conformations. Significance StatementThermodynamic preferences of nucleic acids to adopt non-native conformations are crucial for understanding how they function but prove difficult to measure experimentally. As a result, little is known about how these thermodynamic preferences vary with sequence and structural contexts, physiological conditions, and chemical modifications. Here, we show that modifications stabilizing non-native conformations and rendering them the major state, in conjunction with melting experiments, enable facile measurements of thermodynamic preferences to form various non-native conformations in DNA and RNA. delta-melt provided rare insights into the cooperativity of forming tandem Hoogsteen base pairs and revealed large and distinct sequence-specific preferences to form G-C+ and A-T Hoogsteen and A-T base open conformations in DNA, which may contribute to sequence-specific DNA biochemistry.

biophysics↗

bioRxiv2020

2'-O-methylation alters the RNA secondary structural ensemble

2-O-methyl (Nm) is a highly abundant post-transcriptional RNA modification that plays important biological roles through mechanisms that are not entirely understood. There is evidence that Nm can alter the biological activities of RNAs by biasing the ribose sugar pucker equilibrium toward the C3-endo conformation formed in canonical duplexes. However, little is known about how Nm might more broadly alter the dynamic ensembles of non-canonical RNA motifs. Here, using NMR and the HIV-1 transactivation response (TAR) element as a model system, we show that Nm preferentially stabilizes alternative secondary structures in which the Nm-modified nucleotides are paired, increasing both the abundance and lifetime of a low-populated short-lived excited state by up to 10-fold. The extent of stabilization increased with number of Nm modifications and was also dependent on Mg2+. Through phi ({Phi}) value analysis, the Nm modification also provided rare insights into the structure of the transition state for conformational exchange. Our results suggest that Nm could alter the biological activities of Nm-modified RNAs by modulating their secondary structural ensembles as well as establish the utility of Nm as a tool for the discovery and characterization of RNA excited state conformations.

biochemistry↗

bioRxiv2020

An RNA dynamic ensemble at atomic resolution

Biomolecules do not fold into a single 3D structure but rather form dynamic ensembles of many inter-converting conformations1. Knowledge of dynamic ensembles is key for understanding how biomolecules fold and function, and for rationally manipulating their activities in drug discovery and synthetic biology2-4. However, solving dynamic ensembles of biomolecules at atomic resolution is a major challenge in structural biology because the information required to specify the position of all atoms in thousands of conformations in an ensemble far exceeds the information content of experimental measurements. Here we addressed the data gap and dramatically simplified and accelerated RNA ensemble determination by using structure prediction tools that leverage the growing database of RNA structures to generate a conformational library. Library refinement with NMR residual dipolar couplings enabled determination of an atomic-resolution ensemble for HIV-1 TAR as confirmed by quantum-mechanical calculations of NMR chemical shifts, comparison to a crystal structure of a substate, and through the successful redistribution of the ensemble by design using atomic mutagenesis. The ensemble provides an unprecedented view of how bulge residues cooperatively flip out and undergo sugar repuckering to allow the adjoining helices to stack. The generality of this approach will make determination of atomic-resolution RNA ensembles routine.

biophysics↗

bioRxiv2020

A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolution

Telomeric G-quadruplexes (G4) were long believed to form a protective structure at telomeres, preventing their extension by the ribonucleoprotein telomerase. Contrary to this belief, we have previously demonstrated that parallel-stranded conformations of telomeric G4 can be extended by human and ciliate telomerase. However, a mechanistic understanding of the interaction of telomerase with structured DNA remained elusive. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) microscopy and bulk-phase enzymology to propose a mechanism for the resolution and extension of parallel G4 by telomerase. Binding is initiated by the RNA template of telomerase interacting with the G-quadruplex; nucleotide addition then proceeds to the end of the RNA template. It is only through the large conformational change of translocation following synthesis that the G-quadruplex structure is completely unfolded to a linear product. Surprisingly, parallel G4 stabilization with either small molecule ligands or by chemical modification does not always inhibit G4 unfolding and extension by telomerase. These data reveal that telomerase is a parallel G-quadruplex resolvase.

biochemistry↗