An evolutionary approach to the explanation of origin and development of life infers the implementation of two essential processes: the formation of diversity of some kind of objects and the mechanisms of selection of those which are the most adapted to survival under environmental conditions. The hypothesis of the “RNA world” suggests that RNA was able to store information, reproduce itself and evolve under prebiotic conditions without participation of any enzymes [1
]. Supposedly, recombination events between short oligomers of RNA, formed in polymerization reactions, led to the increase of diversity of nucleotide sequences of RNA. Further development of life demanded an occurrence of mechanisms for molecular selection and evolution. For instance, some studies suggest that montmorillonite could play this role via
absorbing the substrates of recombination reaction in its interlayers [2
]. It is entirely possible that posterior stages of the “RNA world” evolution were signified with substitution of minerals by RNA in the capacity of a template, and complementary interactions became the basis for ancient mechanisms of selection [4
Consecutive cleavage and ligation reactions of RNA are one of the foremost ways leading to their recombination. Both reactions involve transesterification, which implies the involvement of 2′-OH group of ribose in the cleavage site for the ester bond transfer. This is the main reason for most likely participation of RNA rather than DNA in the evolution under prebiotic conditions. Transesterification occurs spontaneously at alkaline pH, and can be accelerated significantly in the presence of organic bases or divalent metal ions, such as Mn2+
, etc. [5
]. Along with a structural role, metal ions promote rearrangement of electronic density in the cleavage or ligation site via
coordination to 2′-, 3′- and 5′- oxygens of the phosphodiester bond [8
]. In chemical terms, ligation reaction is reverse to cleavage; however, RNA fragments undergoing ligation may differ from those formed at the cleavage step, and thereby ensure the formation of novel RNA sequences. Occurrence of such reactions was first reported by Chetverin et al
., who investigated random non-enzymatic recombination reactions in a pool of RNA within molecular colonies, further amplified and detected using Qβ replicase [9
In a previous paper, we reported on the formation of new RNA sequences as a result of consecutive template-directed cleavage and ligation reactions of short RNA oligonucleotides [11
]. In our earlier works with chimeric DNA/RNA oligonucleotides, the chemical mechanism for a new phosphodiester bond formation was investigated. It was shown that reaction intermediates possessed 2′,3′- cyclophosphate and 5′-OH group, and the ligation proceeded through the mechanism of intermolecular transesterification [12
In the present article, we report the studies of consecutive cleavage/ligation reaction of two 96 nts long RNA fragments, catalyzed by Mg2+. The reaction was performed in the presence of complementary oligodeoxyribonucleotide, which governed the recombination within selected RNA sites. The products of recombination were amplified, isolated and their nucleotide sequences were determined. On the basis of sequencing data, the conclusions on the characteristic properties and mechanisms of recombination events were made.
3. Experimental Section
3.1. Enzymes and chemicals
[γ-32P]-ATP (specific activity >3000 Ci/mmol) was from Biosan Co. (Russia). Enzymes: calf intestinal alkaline phosphatase and endonuclease Fok I were purchased from “SibEnzyme”, Russia. T7 RNA polymerase, Taq DNA polymerase, M-MuLV DNA polymerase were produced in this Institute. T4 polynucleotide kinase was from Fermentas. TA cloning was proceeded using “InsTAcloneTM PCR Cloning Kit” (Fermentas). Plasmid DNA was isolated with “GenEluteTM Plasmid Miniprep Kit” (Sigma). BigDye v.1.1 sequencing mixture was from Applied Biosystems. All the chemicals were of analytical or ACS grade, and autoclaved Milli-QTM water was used for all procedures. M2-RNA of influenza virus was synthesized by T7 transcription from plasmid pSVK3M2, this Institute collection. HIV-1 fragment was obtained via transcription of plasmid pHIV-2, kindly provided by Prof. H.J. Gross (University of Wurzburg, Germany).
Oligonucleotides were synthesized using standard automated solid-phase methods and purified by reverse-phase liquid chromatography in this Institute. Primers were as follows: M2–96for – ACAAGCTTTAATACGACTCACTATAGGGCCTTCTACGGAAGGAGTACC (T7 promoter region is underlined), M2–96rev – CGAGACAAAATGACTGTCGTCAGC, Mfor – CTACGGAAGGAGTACC TGA, Mrev – ATGACTGTCGTCAGCATC, Hfor – GAGATCCCTCAGACCACT, M13/pUC_for – GTAAAACGACGGCCAGT, M13/pUC_rev – CAGGAAACAGCTATGAC. ON-template – CGATCCACAGCACTCACCGTCTAGAGTAAAGC (overhangs are underlined).
3.3. 5′-32P-oligonucleotide labeling
Primer Mrev was 5′-end labeled with 32P using T4 polynucleotide kinase and [γ-32P]-ATP. A reaction system (total volume 10 μL) containing 300 pmol of oligonucleotide, ligation buffer, 10 u of polynucleotide kinase and 0.1 mCi [γ-32P]-ATP was incubated at 37 °C for 1 hour. Then labeled oligonucleotide was purified in 20% denaturing polyacrylamide gel (dPAAG).
3.4. Plasmid linearization
Plasmid pHIV-2 was linearized with Fok I endonuclease according to the manufacturer’s manual. Reaction mixture of a total volume 200 μL, containing 40 μg of plasmid DNA, restriction buffer and 120 u of endonuclease, was incubated at 37 °C for 4 hours. Then mixture was extracted consecutively with solution TE-phenol-chloroform (1:1) and chloroform-isoamyl alcohol (24:1). DNA from the aqueous phase was precipitated with ethanol and dissolved in water.
3.5. Amplification of M2-DNA fragment
M2-DNA fragment was amplified from the plasmid pSVK3M2 using primers M2–96for and M2–96rev. PCR mixture contained 67 mM Tris-HCl (pH 8.0), 16 mM (NH4)2SO4, 1.5 mM MgCl2, 0.01% Tween-20, 0.2 mM dNTPs, 1.3 μM each PCR primer, 0.5 μg/mL plasmid and 5 u of Taq DNA polymerase in a final volume of 50 μL. PCR was performed in Hybaid PCR Express thermal cycler, starting with 3 min of preincubation at 95 °C, and followed by 30 amplification cycles (1 min, 94 °C; 1 min, 52 °C; 1 min, 72 °C). Amplification product (121 bp) was purified by phenol extraction (TEphenol : chloroform (1:1), then chloroform only) and precipitated from the aqueous phase with ethanol. Dissolved in water precipitates were used as templates for T7 transcription.
3.6. T7 transcription
We applied T7 transcription for the synthesis of M2-RNA and HIV-RNA from M2-DNA (obtained by amplification of pSVK3M2 plasmid fragment) and linearized plasmid pHIV-2, respectively. Reaction mixtures of total volume 100 μL, containing 40 mM Tris-HCl (pH 8.1), 16 mM MgCl2, 2 mM spermidine, 4 mM NTPs, 5 mM DTT, 0.2 mg/mL BSA, 0.6 mg/mL of DNA and 480 u of T7 polymerase, were incubated at 37 °C for 4 hours. After incubation, RNA was purified by phenol extraction [phenol-chloroform (1:1), then chloroform-isoamyl alcohol (24:1)], followed by purification in 8% native PAAG. The visualized band was cut out and RNA was eluted from the gel with 3 mL of elution solution (0.6 M NH4Ac (pH 5.0), 0.1 mM EDTA, 0.1% SDS) on a shaker at 4 °C for 4 hours. Eluted RNA was precipitated with ethanol and dissolved in water.
3.7. Dephosphorylation of RNA
A reaction mixture (total volume 50 μL) containing 8 μg of M2-RNA or HIV-RNA, 50 mM bis(tris(oxymethyl)methylamino)propane HCl buffer (pH 8.0), 2% formamide, 0.2% SDS, 1.25 mM DTT and 1.5 u of Calf intestinal alkaline phosphatase (CIP) was incubated at 37 °C for 45 minutes. Then 1.5 u of CIP was added again to the reaction mixture and it was additionally incubated under the same conditions. After incubation, mixture was subjected to twofold extraction with phenolchloroform (1:1), then chloroform-isoamyl alcohol (24:1). RNA was precipitated from the aqueous phase by 95% ethanol, washed twice with 75% ethanol, dissolved in water and stored at −20 °C.
3.8. Non-enzymatic cleavage/ligation reaction
Reaction mixtures (total volumes 30 μL) containing bis(tris(oxymethyl)methylamino)propane HCl buffer (pH 7.5, 8.0, 8.5), 0.5 μM dephosphorylated M2-RNA and HIV-RNA, 1.0 μM of the ONtemplate (in template-directed reactions) and 5 mM MgCl2, were incubated at 37 °C for 3 days. After incubation, EDTA was added to the ligation mixtures in a concentration equal to that of MgCl2. Volumes of all probes were adjusted to 100 μL with water, and RNA was precipitated with ethanol in the presence of 0.3 M NaAc and 10 μg of glycogen. Precipitates were washed with ethanol, dissolved in 15 μL of water and used in reverse transcription reaction.
3.9. Reverse transcription and PCR of ligation products
The DNA template for PCR amplification was produced from the ligation mixture of recombinant RNA in reverse transcription reaction. A reaction mixture with an initial volume of 12 μL, containing 1.25 μM of specific reverse primer and 1 μL of mixture of products of the cleavage/ligation reaction (see p. 3.8), was subjected to denaturation at 70 °C for 5 min and then chilled on ice for 3 min. After that NTPs of a final concentration 1 mM and M-MuLV buffer were added, volume was adjusted to 18 μL with water, and mixtures were incubated at 37 °C for 5 min. Finally, 20 u of M-MuLV polymerase were added and the mixture of a final volume 20 μL was incubated at 42 °C for 60 min. The reaction was terminated by heating at 70 °C for 10 min. Obtained cDNA was directly used in PCR. PCR was performed from reverse transcription (RT) products using primers Hfor and Mrev. In a positive control (to confirm M2-RNA presence in the reaction mixture) primers Mfor and Mrev were used. PCR mixtures contained 67 mM Tris-HCl (pH 8.0), 16 mM (NH4)2SO4, 1.5 mM MgCl2, 0.01% Tween-20, 0.25 mM dNTPs, 0.8 μM each PCR primer, 1 μL of RT-product in dilutions 1:1, 1:10−3, 1:10−6 and 2 u of Taq DNA polymerase in a total volume of 20 μL. PCR was started with 3 min of preincubation at 95 °C and followed by 25 cycles of amplification (1 min, 94 °C; 1 min, 56 °C; 1 min, 72 °C). Final cycle contained prolonged elongation step (72 °C, 10 min) for complete formation of sticky A-ends. PCR products were used directly for TA cloning. In parallel, we run PCR with 5′-32P-labeled Mrev primers for visualization purposes. In this case aliquots of PCR products were mixed with loading buffer and applied on 10% dPAAG electrophoresis. Visualization was accomplished by exposing dried gels on FX Imaging Screen, followed by screen scanning on Molecular Imager FX-PRO Plus (Bio-Rad) and images processing in Quantity One v.4.2.3 Software (Bio-Rad). Amplification products were identified by electrophoresis on 10% dPAAG using DNA ladder, obtained via chemical cleavage of M2–80 radioactive PCR product (synthesized using primers Mfor and 5′-32P-Mrev, product length 80 bp) at adenine and guanine sites. Reaction mixture of a volume 30 μL, containing 10 μL of 5′-32P-labeled M2–80, and 20 μL of 3% diphenylamine solution in formic acid, was incubated at 37 °C for 5 and 10 minutes. At the end of incubation, 100 μL of water was added and reaction mixtures were subjected to twofold extraction with 300 μL of diethyl ether. DNA from the aqueous phase was precipitated with 10 volumes of 2% LiClO4 solution in acetone, pellets were washed with acetone, dissolved in water and then added to the loading solution.
3.10. TA cloning
The PCR products were cloned using the “InsTAcloneTM PCR Cloning Kit” (Fermentas), according to the manual provided by the supplier. Briefly, 0.54 pmol of PCR products, possessing sticky A-ends, was ligated with 0.18 pmol of linear plasmid vector pTZ57R/T, containing sticky ddT-ends, using T4 DNA ligase. The obtained recombinant plasmids were used for transformation of competent E. coli DH5α cells, prepared using the same Kit. Transformed cells were plated at LB agar Petri dishes with ampicillin at concentration of 50 μg/mL. Dishes were incubated at 37 °C for 20 hours and then stored at 4 °C. Plasmids were isolated using “GenEluteTM Plasmid Miniprep Kit” (Sigma) according to the manual of supplier. Briefly, night cultures (OD~0.5) were prepared from corresponding bacterial colonies in 3 mL of LB medium containing ampicillin at a concentration of 50 μg/mL. Cells were harvested by centrifugation, resuspended, lysed and then the solution was neutralized. Cell debris was precipitated by centrifugation, cleared lysate was loaded at microcentrifuge spin columns, and after washing plasmid DNA was eluted from the column with 100 μL of water. Isolated plasmids were used directly for sequencing purposes.
3.11. Bacterial colony PCR
Fragments of individual bacterial colonies were dissolved in 20 μL of PCR master mix, containing 67 mM Tris-HCl (pH 8.0), 16 mM (NH4)2SO4, 1.5 mM MgCl2, 0.01% Tween-20, 0.2 mM dNTPs, 0.3 μM of primers M13/pUC_for and M13/pUC_rev, and 2 u of Taq DNA polymerase. PCR was started with 3 min of preincubation at 95 °C and followed by 27 cycles of amplification (30 s, 94 °C; 30 s, 51 °C; 1 min, 72 °C). PCR products were analyzed in 8% dPAAG and visualized by staining of gels with ethidium bromide.
3.12. DNA sequencing
Sequencing of constructs possessing the DNA inserts was performed via Sanger sequencing reaction with mixture BigDye v.1.1 (Applied Biosystems) followed by further analysis of products at automatic gel analyzer ABI 3130xl (Applied Biosystems). For the sequencing reaction, 5 pmol of M13/pUC_rev primer, 200–400 fmol of plasmid, sequencing buffer and 1.5 μL of BigDye were mixed and adjusted to a final volume of 30 μL by adding water. The reaction was run according to the following program: 10 s preincubation at 96 °C; 3 cycles: 8 s, 96 °C; 4 min, 64 °C; 5 cycles: 8 s, 96 °C; 4 min, 60 °C; 17 cycles: 10 s, 96 °C; 5 s, 50 °C; 4 min, 60 °C; final step denaturation: 3 min, 96 °C. Products of sequencing reaction were purified from the unincorporated BigDye by precipitation with isopropyl alcohol, pellets were dried on SpeedVac evaporator for 3 minutes, dissolved in formaldehyde and subjected to analysis at gene analyzer. Sequences were determined in Sequence Scanner Software v. 1.0 (Applied Biosystems) and aligned with initial RNA sequences using Vector NTI v.10.0.1 Software (Invitrogen).
The non-enzymatic spontaneous recombination reactions discussed in this work were perfomed under very simple conditions, and, presumably, could be easily realized in prebiotic proteinless environment and lead to the formation of a pool of new RNA molecules. Notwithstanding the simplicity of chemical conditions, the processes taking place in the reaction system are very complicated and hardly predicted. It was found that nucleic acid template did not direct, but just facilitated the ligation, providing opportunity for approaching RNA dangling ends of different lengths and nucleotide sequences. RNA molecules appeared not to be prone to ligation within a fully complementary complex with a template, though some clones with this type of recombinant product were detected. Instead, the ligation occurred mostly in the elements of imperfect secondary structure of RNA, namely bulge loops of a size of one or three nucleotides and internal loops of a two or three nucleotide size. In spite of energetic preference of ligation within a duplex with the template, the predominant part of new phosphodiester bonds is formed in the 3 nts RNA bulges that strain spatial structure of RNA due to the double helix bend in the bulge area. However, local conformational preference for formation of this sort of products may be prevalent over thermodynamic stability of newly formed structures, providing an opportunity for occurrence of loop-bearing RNAs with novel sequences. Along with the products of template-governed ligation, we observed a number of products of template-independent ligation. However, formation of this sort of molecules may be attributed to the action of RNA substrates as templates for their self-ligation. If this is the case, proposed non-enzymatic mechanisms seem as a very plausible pathway for the formation, selection and evolution of first informational biopolymers in the prebiotic world.