Search

The non-enzymatic replication of the primordial genetic material is thought to have enabled the evolution of early forms of RNA-based life. However, the replication of oligonucleotides long enough to encode catalytic functions is problematic due to the low efficiency of template copying with mononucleotides. We show that template-directed ligation can assemble long RNAs from shorter oligonucleotides, which would be easier to replicate. 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 enable the copying of RNA templates by the multistep ligation of tetranucleotide building blocks, as well as the assembly of long oligonucleotides using short splint oligonucleotides. We also demonstrate the formation of long oligonucleotides inside model prebiotic vesicles, which suggests a potential route to the assembly of artificial cells capable of evolution., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Non-enzymatic RNA replication may have been one of the processes involved in the appearance of life on Earth. Attempts to recreate this process in a laboratory setting have not been successful thus far, highlighting a critical need for finding prebiotic conditions that increase the rate and the yield. Now a highly parallel assay for template directed RNA synthesis is presented that relies on the intrinsic fluorescence of a 2-aminopurine modified G-quadruplex. The application of the assay to examine the combined influence of multiple variables including pH, divalent metal concentrations and ribonucleotide concentrations on the copying of RNA sequences is demonstrated. The assay enables a direct survey of physical and chemical conditions, potentially prebiotic, which could enable the chemical replication of RNA., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Oligonucleotides containing an alkylene intrastrand cross-link (IaCL) between the O(6) -atoms of two consecutive 2'-deoxyguanosines (dG) were prepared by solid-phase synthesis. UV thermal denaturation studies of duplexes containing butylene and heptylene IaCL revealed a 20 °C reduction in stability compared to the unmodified duplexes. Circular dichroism profiles of these IaCL DNA duplexes exhibited signatures consistent with B-form DNA. Human O(6) -alkylguanine DNA alkyltransferase (hAGT) was capable of repairing both IaCL containing duplexes with slightly greater efficiency towards the heptylene analog. Interestingly, repair efficiencies of hAGT towards these IaCL were lower compared to O(6) -alkylene linked IaCL lacking the 5'-3'-phosphodiester linkage between the connected 2'-deoxyguanosine residues. These results demonstrate that the proficiency of hAGT activity towards IaCL at the O(6) -atom of dG is influenced by the backbone phosphodiester linkage between the cross-linked residues., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Oligonucleotides tethered by an alkylene linkage between the O(6) -atoms of two consecutive 2'-deoxyguanosines, which lack a phosphodiester linkage between these residues, have been synthesized as a model system of intrastrand cross-linked (IaCL) DNA. UV thermal denaturation studies of duplexes formed between these butylene- and heptylene-linked oligonucleotides with their complementary DNA sequences revealed about 20 °C reduction in stability relative to the unmodified duplex. Circular dichroism spectra of the model IaCL duplexes displayed a signature characteristic of B-form DNA, suggesting minimal global perturbations are induced by the lesion. The model IaCL containing duplexes were investigated as substrates of O(6) -alkylguanine DNA alkyltransferase (AGT) proteins from human and E. coli (Ada-C and OGT). Human AGT was found to repair both model IaCL duplexes with greater efficiency towards the heptylene versus butylene analog adding to our knowledge of substrates this protein can repair., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The bisalkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), used in cancer chemotherapy to hinder cellular proliferation, forms lethal interstrand cross-links (ICLs) in DNA. BCNU generates an ethylene linkage connecting the two DNA strands at the N1 atom of 2'-deoxyguanosine and N3 atom of 2'-deoxycytidine, which is a synthetically challenging probe to prepare. To this end, an ICL duplex linking the N1 atom of 2'-deoxyinosine to the N3 atom of thymidine via an ethylene linker was devised as a mimic. We have solved the structure of this ICL duplex by a combination of molecular dynamics and high-field NMR experiments. The ethylene linker is well-accommodated in the duplex with minimal global and local perturbations relative to the unmodified duplex. These results may account for the substantial stabilization of the ICL duplex observed by UV thermal denaturation experiments and provides structural insights of a probe that may be useful for DNA repair studies., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)