Szostak, J. | Search bioRxiv

bioRxiv2024

RNA complexes with nicks and gaps: thermodynamic and kinetic effects of coaxial stacking and dangling ends

Multiple RNA strands can interact in solution and assume a large variety of configurations dictated by their potential for base pairing. Although duplex formation from two complementary oligonucleotides has been studied in detail, we still lack a systematic characterization of the behavior of higher order complexes. Here we focus on the thermodynamic and kinetic effects of an upstream oligonucleotide on the binding of a downstream oligonucleotide to a common template, as we vary the sequence and structure of the contact interface. We show that coaxial stacking in RNA is well correlated with but much more stabilizing than helix propagation over an analogous intact double helix step (median {Delta} {Delta} G37{degrees}C {approx} 1.7 kcal/mol). Consequently, approximating coaxial stacking in RNA with the helix propagation term leads to large discrepancies between predictions and our experimentally determined melting temperatures, with an offset of {approx} 10{degrees}C. Our kinetic study reveals that the hybridization of the downstream probe oligonucleotide is impaired (lower kon) by the presence of the upstream oligonucleotide, with the thermodynamic stabilization coming entirely from an extended lifetime (lower koff) of the bound downstream oligonucleotide, which can increase from seconds to months. Surprisingly, we show that the effect of nicks is dependent on the length of the stacking oligonucleotides, and we discuss the binding of ultrashort (1 [~] 4 nt) oligonucleotides that are relevant in the context of the origin of life. The thermodynamic and kinetic data obtained in this work allow for the prediction of the formation and stability of higher order multi-stranded complexes. Graphic entry for the Table of Contents (TOC) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/589350v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@8b11d3org.highwire.dtl.DTLVardef@3f55c6org.highwire.dtl.DTLVardef@185703org.highwire.dtl.DTLVardef@1f8a9e8_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2019

Assembly of a functional ribozyme from short oligomers by enhanced non-enzymatic ligation

The non-enzymatic replication of the primordial genetic material is thought to have enabled the evolution of the first ribozymes, leading to early forms of RNA-based life. However, the reported rate of chemical RNA ligation is extremely slow. Here we show that the rate of ligation can be greatly enhanced by employing a 3'-amino group at the 3'-end of each oligonucleotide, in combination with an N-alkyl imidazole organocatalyst. These modifications allow the rapid copying of long RNA templates by multi-step ligation of tetranucleotides, as well as the assembly of long oligonucleotides from short template splints. Our work shows that a functional RNA ligase ribozyme can be assembled from relatively short oligonucleotides, demonstrating a transition from non-enzymatic ligation to enzymatic ligation. We suggest that the genomes of primitive protocells could have consisted of relatively easily replicated oligonucleotides as short as 10 to 12 nucleotides in length.

synthetic biology↗