Salt bridge dynamics in nonspecific protein/DNA binding: a comparative analysis of Elk1 and ETV6

Electrostatic protein/DNA interactions arise from the neutralization of the DNA phosphodiester backbone as well as coupled exchanges by charged protein residues as salt bridges or with mobile ions. Much focus...

Polyamines Mediate Folding of Primordial Hyper-Acidic Helical Proteins

Polyamines are known to mediate diverse biological processes, and specifically to bind and stabilize compact conformations of nucleic acids, acting as chemical chaperones that promote folding by offsetting the repulsive negative charges of the phosphodiester backbone. However, whether and how polyamines modulate the structure and function of proteins remains unclear. Further, early proteins are thought to have been highly acidic, like nucleic acids, due to a scarcity of basic amino acids in the prebiotic context. Perhaps polyamines, the abiotic synthesis of which is simple, could have served as chemical chaperones for such primordial proteins? We replaced all lysines of an ancestral 60-residue helix-bundle protein to glutamate, resulting in a disordered protein with 21 glutamates in total. Polyamines efficiently induce folding of this hyper-acidic protein at sub-millimolar concentrations, and their potency scaled with the number of amine groups. Compared to cations, polyamines were several orders of magnitude more potent than Na+, while Mg2+ and Ca2+ had an effect similar to a di-amine, inducing folding at approximately seawater concentrations. We propose that (i) polyamines and dications may have had a role in promoting folding of early proteins devoid of basic residues, and that (ii) coil-helix transitions could be the basis of polyamine regulation in contemporary proteins.

Understanding the effect of controlling phosphorothioate chirality in the DNA gap on the potency and safety of gapmer antisense oligonucleotides

Therapeutic oligonucleotides are often modified using the phosphorothioate (PS) backbone modification which enhances stability from nuclease mediated degradation. However, substituting oxygen in the phosphodiester backbone with sulfur introduce chirality into the backbone such that a full PS 16-mer oligonucleotide is comprised of 215 distinct stereoisomers. As a result, the role of PS chirality on the performance of antisense oligonucleotides (ASOs) has been a subject of debate for over two decades. We carried out a systematic analysis to determine if controlling PS chirality in the DNA gap region can enhance the potency and safety of gapmer ASOs modified with high-affinity constrained Ethyl (cEt) nucleotides in the flanks. As part of this effort, we examined the effect of systematically controlling PS chirality on RNase H1 cleavage patterns, protein mislocalization phenotypes, activity and toxicity in cells and in mice. We found that while controlling PS chirality can dramatically modulate interactions with RNase H1 as evidenced by changes in RNA cleavage patterns, these were insufficient to improve the overall therapeutic profile. We also found that controlling PS chirality of only two PS linkages in the DNA gap was sufficient to modulate RNase H1 cleavage patterns and combining these designs with simple modifications such as 2′-OMe to the DNA gap resulted in dramatic improvements in therapeutic index. However, we were unable to demonstrate improved potency relative to the stereorandom parent ASO or improved safety over the 2′-OMe gap-modified stereorandom parent ASO. Overall, our work shows that while controlling PS chirality can modulate RNase H1 cleavage patterns, ASO sequence and design are the primary drivers which determine the pharmacological and toxicological properties of gapmer ASOs.

Prebiotically Plausible ‘Patching’ of RNA Backbone Cleavage Through a 3′-5′ Pyrophosphate Linkage

ABSTRACTAchieving multiple cycles of RNA replication within a model protocell would be a critical step towards demonstrating a path from prebiotic chemistry to cellular biology. Any model for early life based on an ‘RNA world’ must account for RNA strand cleavage and hydrolysis, which would degrade primitive genetic information and lead to an accumulation of truncated, phosphate-terminated strands. We show here that cleavage of the phosphodiester backbone is not an endpoint for RNA replication. Instead, 3′ -phosphate terminated RNA strands are able to participate in template-directed copying reactions with activated ribonucleotide monomers. These reactions form a pyrophosphate linkage, the stability of which we have characterized in the context of RNA copying chemistry. We found that the pyrophosphate bond is relatively stable within an RNA duplex and in the presence of chelated magnesium. Under these conditions, pyrophosphate-RNA can act as a temporary ‘patch’ to template the polymerization of canonical ribonucleotides, suggesting a plausible non-enzymatic pathway for the salvage and recovery of genetic information following strand cleavage.

The effect of adenine protonation on RNA phosphodiester backbone bond cleavage elucidated by deaza-nucleobase modifications and mass spectrometry

The catalytic strategies of small self-cleaving ribozymes often involve interactions between nucleobases and the ribonucleic acid (RNA) backbone. Here we show that multiply protonated, gaseous RNA has an intrinsic preference for the formation of ionic hydrogen bonds between adenine protonated at N3 and the phosphodiester backbone moiety on its 5′-side that facilitates preferential phosphodiester backbone bond cleavage upon vibrational excitation by low-energy collisionally activated dissociation. Removal of the basic N3 site by deaza-modification of adenine was found to abrogate preferential phosphodiester backbone bond cleavage. No such effects were observed for N1 or N7 of adenine. Importantly, we found that the pH of the solution used for generation of the multiply protonated, gaseous RNA ions by electrospray ionization affects phosphodiester backbone bond cleavage next to adenine, which implies that the protonation patterns in solution are at least in part preserved during and after transfer into the gas phase. Our study suggests that interactions between protonated adenine and phosphodiester moieties of RNA may play a more important mechanistic role in biological processes than considered until now.

Hatchet ribozyme structure and implications for cleavage mechanism

Small self-cleaving ribozymes catalyze site-specific cleavage of their own phosphodiester backbone with implications for viral genome replication, pre-mRNA processing, and alternative splicing. We report on the 2.1-Å crystal structure of the hatchet ribozyme product, which adopts a compact pseudosymmetric dimeric scaffold, with each monomer stabilized by long-range interactions involving highly conserved nucleotides brought into close proximity of the scissile phosphate. Strikingly, the catalytic pocket contains a cavity capable of accommodating both the modeled scissile phosphate and its flanking 5′ nucleoside. The resulting modeled precatalytic conformation incorporates a splayed-apart alignment at the scissile phosphate, thereby providing structure-based insights into the in-line cleavage mechanism. We identify a guanine lining the catalytic pocket positioned to contribute to cleavage chemistry. The functional relevance of structure-based insights into hatchet ribozyme catalysis is strongly supported by cleavage assays monitoring the impact of selected nucleobase and atom-specific mutations on ribozyme activity.

Decoding on the ribosome depends on the structure of the mRNA phosphodiester backbone

During translation, the ribosome plays an active role in ensuring that mRNA is decoded accurately and rapidly. Recently, biochemical studies have also implicated certain accessory factors in maintaining decoding accuracy. However, it is currently unclear whether the mRNA itself plays an active role in the process beyond its ability to base pair with the tRNA. Structural studies revealed that the mRNA kinks at the interface of the P and A sites. A magnesium ion appears to stabilize this structure through electrostatic interactions with the phosphodiester backbone of the mRNA. Here we examined the role of the kink structure on decoding using a well-defined in vitro translation system. Disruption of the kink structure through site-specific phosphorothioate modification resulted in an acute hyperaccurate phenotype. We measured rates of peptidyl transfer for near-cognate tRNAs that were severely diminished and in some instances were almost 100-fold slower than unmodified mRNAs. In contrast to peptidyl transfer, the modifications had little effect on GTP hydrolysis by elongation factor thermal unstable (EF-Tu), suggesting that only the proofreading phase of tRNA selection depends critically on the kink structure. Although the modifications appear to have no effect on typical cognate interactions, peptidyl transfer for a tRNA that uses atypical base pairing is compromised. These observations suggest that the kink structure is important for decoding in the absence of Watson–Crick or G–U wobble base pairing at the third position. Our findings provide evidence for a previously unappreciated role for the mRNA backbone in ensuring uniform decoding of the genetic code.