Effect of Molecular Crowding on DNA Polymerase Reactions along Unnatural DNA Templates

Takahashi, Shuntaro; Herdewijn, Piet; Sugimoto, Naoki

doi:10.3390/molecules25184120

Cited by 6 publications

(6 citation statements)

References 24 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Enhancement of ribozyme polymerase activity at low millimolar Mg 2+ underscores the generality of the beneficial effects of crowded environments on ribozyme-catalyzed RNA assembly. Interestingly, molecular crowding has also been found to enhance the polymerization of NTPs and dNTPs by biologically derived protein polymerases, which further supports the role of crowding in facilitating nucleic acid assembly.…”

Section: Resultsmentioning

confidence: 66%

Molecular Crowding Facilitates Ribozyme-Catalyzed RNA Assembly

2023

View full text Add to dashboard Cite

Catalytic RNAs or ribozymes are considered to be central to primordial biology. Most ribozymes require moderate to high concentrations of divalent cations such as Mg2+ to fold into their catalytically competent structures and perform catalysis. However, undesirable effects of Mg2+ such as hydrolysis of reactive RNA building blocks and degradation of RNA structures are likely to undermine its beneficial roles in ribozyme catalysis. Further, prebiotic cell-like compartments bounded by fatty acid membranes are destabilized in the presence of Mg2+, making ribozyme function inside prebiotically relevant protocells a significant challenge. Therefore, we sought to identify conditions that would enable ribozymes to retain activity at low concentrations of Mg2+. Inspired by the ability of ribozymes to function inside crowded cellular environments with <1 mM free Mg2+, we tested molecular crowding as a potential mechanism to lower the Mg2+ concentration required for ribozyme-catalyzed RNA assembly. Here, we show that the ribozyme-catalyzed ligation of phosphorimidazolide RNA substrates is significantly enhanced in the presence of the artificial crowding agent polyethylene glycol. We also found that molecular crowding preserves ligase activity under denaturing conditions such as alkaline pH and the presence of urea. Additionally, we show that crowding-induced stimulation of RNA-catalyzed RNA assembly is not limited to phosphorimidazolide ligation but extends to the RNA-catalyzed polymerization of nucleoside triphosphates. RNA-catalyzed RNA ligation is also stimulated by the presence of prebiotically relevant small molecules such as ethylene glycol, ribose, and amino acids, consistent with a role for molecular crowding in primordial ribozyme function and more generally in the emergence of RNA-based cellular life.

show abstract

Section: Resultsmentioning

confidence: 66%

Molecular Crowding Facilitates Ribozyme-Catalyzed RNA Assembly

2023

View full text Add to dashboard Cite

show abstract

“…This enhancement of ribozyme polymerase activity at low millimolar Mg 2+ underscores the generality of the beneficial effects of crowded environments on ribozyme-catalyzed RNA assembly. Interestingly, molecular crowding has also been found to enhance the polymerization of NTPs 33 and dNTPs 34 by biologically-derived protein polymerases, which further supports the role of crowding in facilitating nucleic acid assembly.…”

Section: Crowding Stimulates Rna-catalyzed Rna Polymerization At Low ...mentioning

confidence: 62%

Molecular crowding facilitates ribozyme-catalyzed RNA assembly

DasGupta

Zhang

Szostak

2023

Preprint

View full text Add to dashboard Cite

Catalytic RNAs or ribozymes are considered to be central to primordial biology. Most ribozymes require moderate to high concentrations of divalent cations such as Mg2+ to fold into their catalytically competent structures and perform catalysis. However, undesirable effects of Mg2+ such as hydrolysis of reactive RNA building blocks and degradation of RNA structures are likely to undermine its beneficial roles in ribozyme catalysis. Further, prebiotic cell-like compartments bounded by fatty acid membranes are destabilized in the presence of Mg2+, making ribozyme function inside prebiotically relevant protocells a significant challenge. Therefore, we sought to identify conditions that would enable ribozymes to retain activity in low concentrations of Mg2+. Inspired by the ability of ribozymes to function inside crowded cellular environments with <1 mM free Mg2+, we tested molecular crowding as a potential mechanism to lower the Mg2+ concentration required for ribozyme-catalyzed RNA assembly. Here, we show that the ribozyme-catalyzed ligation of phosphorimidazolide RNA substrates is significantly enhanced in the presence of the artificial crowding agent polyethylene glycol. We also found that molecular crowding preserves ligase activity under denaturing conditions such as alkaline pH and the presence of urea. We also show that crowding-induced stimulation of RNA-catalyzed RNA assembly is not limited to phosphorimidazolide ligation but extends to the RNA-catalyzed polymerization of nucleoside triphosphates. RNA-catalyzed RNA ligation is also stimulated by the presence of prebiotically relevant small molecules such as ethylene glycol, ribose, and amino acids, consistent with a role for molecular crowding in primordial ribozyme function and more generally, in the emergence of RNA-based cellular life.

show abstract

“…As discussed above, for the 50 base pairs chain, only a few base pairs (10)(11)(12)(13)(14)(15) open initially (the reason for the first smaller peak). The major section of the chain opens nearly in a narrow temperature range.…”

Section: Dna In the Presence Of More Than One Crowdermentioning

confidence: 81%

“…The molecular crowders play a crucial role in phase separation in the cytoplasm and condensation of DNA into the nucleoid of bacterial cells [11]. Recently, Takanashi has shown how the molecular crowders modify the DNA and RNA polymerase reactions [12,13]. In another interesting study, Ghosh et al investigated the stability of short DNA duplexes in different solutes using the nearest neighbour (NN) model [1].…”

Section: Introductionmentioning

confidence: 99%

The crowding effect on the melting of short DNA: Comparison with experiments

Mathur¹,

Singh²,

Singh³

2022

Preprint

View full text Add to dashboard Cite

We study the effect of crowders on the melting profile of homogeneous and heterogeneous DNA molecules. We find out the melting profile of short DNA molecules and compare our findings with the experiments. We consider some random distribution of crowders along the chain, and by finding out the best match with the experiments, we attempt to identify the location of crowders in the experimental findings of Ghosh [1]. We also study the melting of homogeneous DNA molecules of different lengths (25, 50, 75) in the presence of only one crowder in the chain. By varying the location of the crowder from one end to the other, we find that the melting temperature is susceptible to the location of the crowder at the ends. At the same time, there is minimal effect on the melting temperature due to the location of the crowder. In vivo, the strength of a crowders may vary along the chain. We study the melting of long heterogeneous chain in presence of five crowders of different strength. We find that there is a significant variation in the melting process of DNA in presence of crowders of variable strength.

show abstract

Effect of Molecular Crowding on DNA Polymerase Reactions along Unnatural DNA Templates

Cited by 6 publications

References 24 publications

Molecular Crowding Facilitates Ribozyme-Catalyzed RNA Assembly

Molecular Crowding Facilitates Ribozyme-Catalyzed RNA Assembly

Molecular crowding facilitates ribozyme-catalyzed RNA assembly

The crowding effect on the melting of short DNA: Comparison with experiments

Contact Info

Product

Resources

About