Non-enzymatic template-directed recombination of RNAs

Abstract

RNA non-enzymatic recombination reactions are of great interest within the hypothesis of the "RNA world", which argues that at some stage of prebiotic life development proteins were not yet engaged in biochemical reactions and RNA carried out both the information storage task and the full range of catalytic roles necessary in primitive self-replicating systems. Here we report on the study of recombination reaction occuring between two 96 nucleotides (nts) fragments of RNAs under physiological conditions and governed by a short oligodeoxyribonucleotide template, partially complementary to sequences within each of the RNAs. Analysis of recombination products shows that ligation is predominantly template-directed, and occurs within the complementary complex with the template in "butt-to-butt" manner, in 1- or 3- nts bulges or in 2-3 nts internal loops. Minor recombination products formed in the template-independent manner are detected as well.

Keywords: RNA; RNA bulge loops; RNA internal loops; RNA world; non-enzymatic ligation; non-enzymatic recombination; origin of life.

Figure 1.

Fragments of HIV-RNA and M2-RNA, used as substrates in the non-enzymatic cleavage/ligation (recombination) reaction. Regions of RNAs, complementary to the oligodeoxyribonucleotide template, are highlighted in red. Arrows show internucleotide linkages most susceptible to cleavage (“fragile” sites) [13,16].

Figure 2.

Template-directed non-enzymatic cleavage/ligation (recombination) reaction. 1) Formation of a pseudoduplex between HIV-RNA (left-hand substrate), M2-RNA (right-hand substrate) and the ON-template. 2) Cleavage of HIV-RNA and M2-RNA at “fragile” sites outside the pseudoduplex RNAs:template, with formation of terminal 2′,3′-cyclophosphates and 5′-OH. 3) and 4) Ligation within complementary complex of RNAs with the template in the presence of Mg²⁺. Formation of new phosphodiester bond between the fragments of HIV-RNA and M2-RNA. Arrows show the sequence of processes taking place in the reaction system.

Figure 3.

First step of identification of recombinant RNAs. A) Reverse transcription reaction with a primer M_rev. B) PCR with primers H_for and M_rev.

Figure 4.

RT-PCR analysis of the products of template-directed recombination of M2- RNA and HIV-RNA. Autoradiogram of a 10% denaturing PAAG. “Incubated” group of lanes: RT-PCR analysis of reaction mixtures incubated under conditions of cleavage/ligation reaction at 37 °C for 3 days. 1) positive control (PCR with primers M_for and M_rev); 2), 3), 4): PCR with primers H_for and M_rev, using dilutions of cDNA mixture 1:1, 1:10⁻³ and 1:10⁻⁶, respectively. “NI” group: reaction mixture without incubation was used for RT-PCR analysis. 1) positive control (PCR with primers M_for and M_rev); 2) PCR with primers H_for and M_rev, and non-diluted cDNA mixture. “Control” group: RT-PCR was performed in the absence of RNA (1) or in the absence of cDNA (2 and 3) with PCR primers H_for and M_rev (1 and 2) and M_for and M_rev (3). “L”: DNA ladder (product of partial hydrolysis of M2–80 PCR product at adenine and guanine sites). Location of major products in gel is marked with arrows. M_rev primer was 5′-³²P-labelled in all reaction mixtures.

Figure 5.

Bacterial colonies screening. Representative electrophoresis showing all detected types of products of RNA recombination. Lanes 1–15: products of bacterial colony PCR, amplified with the use of primers M13/pUC_for and M13/pUC_rev. C1 – positive control (PCR from circular plasmid without TA-insert), C2 – negative control (PCR without template), L – double-stranded DNA ladder. Figures below lane numbers show the number of colonies (clones) containing each type of TA-insert.

Figure 6.

Products of template-governed recombination of RNAs. Ligation takes place within a full complementary complex of RNAs with a template (in butt-to-butt manner, P1), in RNA bulges of a size 1 or 3 nucleotides (P2, P3), in symmetric (P13) and asymmetric (P4) internal loops. Fragments of HIV-RNA are shown in red, of M2-RNA—in blue, ON-template is shown in black.