Loopstructures in synthetic oligonucleotides. Hairpin stability and structure studied as a function of loop elongation

The formation of hairpin structures in the homologous, (partly) self-complementary DNA fragments d(ATCCTAT_nTAGGAT), n = 0–7, was studied by means of nuclear magnetic resonance, T-jump and ultra-violet techniques. It is shown that all compounds in the series may adopt hairpin-like conformations, albeit for n < 3 this only occurs to a significant amount at relatively low concentrations (∼ 10μM). For the present series of oligonucleotides, hairpin formation is accompanied by an apparent loop enthalpy significantly different from zero. The stability of the DNA hairpins turns out to be at its maximum for loop lengths of four or five residues, whereas earlier experiments (Tinocoet al., 1973) indicated that loop lengths of six to seven residues are most favourable for RNA hairpins. This is explained by considering the difference in geometry of A-RNA and B-DNA helices.     

Evidence for a four-strand exchange catalyzed by the RecA protein

Strand exchange between two duplexes is usually initiated as a three-strand event that requires the presence of a single-stranded overhang or gap in one of the two molecules. Here we show that the RecA protein can catalyze a four-strand exchange. Specifically, it can recombine short hairpin substrates with homologous stems provided that one of the hairpins possesses a chimeric DNA/RNA backbone. This four-strand exchange reaction goes to completion in the presence of ATPgammaS and releases a stable heteroduplex upon removal of the RecA protein. Under identical conditions, strand exchange between two DNA hairpins is incomplete and generates a nascent heteroduplex that rapidly dissociates when the RecA protein is denatured. Since presynaptic filament formation does not appear to melt either type of hairpin, we propose that exchange occurs between homologously aligned duplexes that are extended and unwound within a RecA filament. The first reaction provides a mechanism for gene targeting by chimeric double-hairpin oligonucleotides while the second reaction explains the ability of the RecA protein to transiently align double-stranded DNA molecules.     

The DNA strand of chimeric RNA/DNA oligonucleotides can direct gene repair/conversion activity in mammalian and plant cell-free extracts

Chimeric oligonucleotides (chimeras), consisting of RNA and DNA bases folded by complementarity into a double hairpin conformation, have been shown to alter or repair single bases in plant and animal genomes. An uninterrupted stretch of DNA bases within the chimera is known to be active in the sequence alteration while RNA residues aid in complex stability. In this study, the two strands were separated in the hope of defining the role each plays in conversion. Using a series of single-stranded oligonucleotides, comprised of all RNA or DNA residues and various mixtures, several new structures have emerged as viable molecules in nucleotide conversion. When extracts from mammalian and plant cells and a genetic readout assay in bacteria are used, single-stranded oligonucleotides, containing a defined number of thioate backbone modifications, were found to be more active than the original chimera structure in the process of gene repair. Single-stranded oligonucleotides containing fully modified backbones were found to have low repair activity and in fact induce mutation. Molecules containing various lengths of modified RNA bases (2′-O-methyl) were also found to possess low activity. Taken together, these results confirm the directionality of nucleotide conversion by the DNA strand of the chimera and further present a novel, modified single-stranded DNA molecule that directs conversion in plant and animal cell-free extracts.     

The synaptic complex of RecA protein participates in hybridization and inverse strand exchange reactions

RecA protein catalyzes strand exchange between homologous single-stranded and double-stranded DNAs. In the presence of ATPgammaS, the post-strand exchange synaptic complex is a stable end product that can be studied. Here we ask whether such complexes can hybridize to or exchange with DNA, 2'-OMe RNA, PNA, or LNA oligonucleotides. Using a gel mobility shift assay, we show that the displaced strand of a 45 bp synaptic complex can hybridize to complementary oligonucleotides with different backbones to form a four-stranded (double D-loop) joint that survives removal of the RecA protein. This hybridization reaction, which confirms the single-stranded character of the displaced strand in a synaptic complex, might initiate recombination-dependent DNA replication if it occurs in vivo. We also show that either strand of the heteroduplex in a 30 bp synaptic complex can be replaced with a homologous DNA oligonucleotide in a strand exchange reaction that is mediated by the RecA filament. Consistent with the important role that deoxyribose plays in strand exchange, oligonucleotides with non-DNA backbones did not participate in this reaction. The hybridization and strand exchange reactions reported here demonstrate that short synaptic complexes are dynamic structures even in the presence of ATPgammaS.     

Complexes of DNA hairpins and a single-stranded oligonucleotide detected by affinity chromatography and mung bean nuclease cleavage

The emergence of a simple translation device consisting of an assembler strand (primordial mRNA) and RNA hairpins (primordial tRNA) is presumed to be an important step leading to the origin of life. The assumption of a non-enzymatic interaction of primordial tRNA and mRNA is experimentally approached. DNA hairpins containing five or more adenosine residues in the loop are able to bind to complementary oligonucleotides covalently bound to cellulose. The exact number of base pairs formed between the hairpins and the assembler strand is determined by two methods applied to DNA hairpin/assembler complexes. The melting temperature of a complex is measured and the cleavage pattern by nuclease from mung bean is determined. The loop of the smallest hairpin able to bind consists of five adenosine residues and only three base pairs are formed. This supports the idea of a primordial recognition similar to the contemporary codon-anticodon interaction.     

Transfection of heteroduplexes containing uracil · guanine or thymine · guanine mispairs into plant cells

We have compared the fate of U · G mispairs or analogous T · G mispairs in DNA heteroduplexes transfected into tobacco protoplasts. The heteroduplex DNA consisted of tomato golden mosaic virus DNA sequences in theEscherichia coli vectors pUC118 or pUC119. After transfection, the mismatched U residues were lost with an efficiency of greater than 95%, probably as a result of the uracil-DNA glycosylase pathway for excision of U residues in any sequence context. In contrast to the preferential removal of the mispaired U residues, biased removal of T residues from analogous heteroduplexes was not seen in the transfected plant cells. Also, we investigated the effect of extensively methylating one strand of the heteroduplex DNA used for transfection. Surprisingly, such methylation resulted in highly biased loss of the mismatched base from the 5-methylcytosine-rich strand of T · G-containing heteroduplexes.Deceased. We dedicate this paper to the memory of this young scientist.     

RNA facilitates RecA-mediated DNA pairing and strand transfer between molecules bearing limited regions of homology

The RecA protein ofEscherichia coli catalyzes homologous pairing and strand exchange between a wide range of molecules showing nucleotide sequence complementarity, including a linear duplex and a single-stranded DNA molecule. We demonstrate that RecA can promote formation of joint molecules when the duplex contains an RNA/DNA hairpin and a single-stranded circle serves as the pairing partner. A chimeric RNA/DNA hairpin can be used to form stable joint molecules with as little as 15 bases of shared homology as long as the RNA stretch contains complementarity to the circle. The joint molecule bears some resemblance to a triple helical structure composed of RNA residues surrounded by two DNA strands which are in a parallel orientation. Evidence is presented that supports the notion that short stretches of RNA can be used in homologous pairing reactions at lengths below that required for DNA-DNA heteroduplex formation.     

The Path from the RNA World

We describe a sequential (step by step) Darwinian model for the evolution of life from the late stages of the RNA world through to the emergence of eukaryotes and prokaryotes. The starting point is our model, derived from current RNA activity, of the RNA world just prior to the advent of genetically-encoded protein synthesis. By focusing on the function of the protoribosome we develop a plausible model for the evolution of a protein-synthesizing ribosome from a high-fidelity RNA polymerase that incorporated triplets of oligonucleotides. With the standard assumption that during the evolution of enzymatic activity, catalysis is transferred from RNA → RNP → protein, the first proteins in the ``breakthrough organism' (the first to have encoded protein synthesis) would be nonspecific chaperone-like proteins rather than catalytic. Moreover, because some RNA molecules that pre-date protein synthesis under this model now occur as introns in some of the very earliest proteins, the model predicts these particular introns are older than the exons surrounding them, the ``introns-first' theory. Many features of the model for the genome organization in the final RNA world ribo-organism are more prevalent in the eukaryotic genome and we suggest that the prokaryotic genome organization (a single, circular genome with one center of replication) was derived from a ``eukaryotic-like' genome organization (a fragmented linear genome with multiple centers of replication). The steps from the proposed ribo-organism RNA genome → eukaryotic-like DNA genome → prokaryotic-like DNA genome are all relatively straightforward, whereas the transition prokaryotic-like genome → eukaryotic-like genome appears impossible under a Darwinian mechanism of evolution, given the assumption of the transition RNA → RNP → protein. A likely molecular mechanism, ``plasmid transfer,' is available for the origin of prokaryotic-type genomes from an eukaryotic-like architecture. Under this model prokaryotes are considered specialized and derived with reduced dependence on ssRNA biochemistry. A functional explanation is that prokaryote ancestors underwent selection for thermophily (high temperature) and/or for rapid reproduction (r selection) at least once in their history. Received: 14 January 1997 / Accepted: 19 May 1997     

Recognition of base pair inversions in duplex by chimeric (alpha,beta) triplex-forming oligonucleotides

DNA recognition by triplex-forming oligonucleotides (TFOs) is usually limited by homopurine-homopyrimidine sequence in duplexes. Modifications of the third strand may overcome this limitation. Chimeric alpha-beta TFOs are expected to form triplex DNA upon binding to non-regular sequence duplexes. In the present study we describe binding properties of chimeric alpha-beta oligodeoxynucleotides in the respect to short DNA duplexes with one, three, and five base pair inversions. Non-natural chimeric TFO's contained alpha-thymidine residues inside (GT) or (GA) core sequences. Modified residues were addressed to AT/TA inversions in duplexes. It was found in the non-denaturing gel-electrophoresis experiments that single or five adjacent base pair inversions in duplexes may be recognized by chimeric alpha-beta TFO's at 10 degrees C and pH 7.8. Three dispersed base pair inversions in the double stranded DNA prevented triplex formation by either (GT) or (GA) chimeras. Estimation of thermal stability of chimeric alpha-beta triplexes showed decrease in T(m) values as compared with unmodified complexes.     

Characterization of an abundant short interspersed nuclear element (SINE) present in Canis familiaris

A short interspersed nuclear element (Can SINE) of ∼130–150 bp was cloned and characterized from Canis familiaris. We demonstrate that this element is interspersed, present approximately every 5–8.3 kbp, and many are sufficiently close to allow IRS (interspersed repetitive DNA sequences) PCR. Sequence analysis of >20 Can SINEs from the dog has identified a conserved region that was used to design oligonucleotides for IRS PCR. Since Can SINEs are not present in human or rodent genomes, IRS PCR using oligonucleotides directed to the conserved region of Can SINEs can be used to simplify analysis of canid DNA in somatic cell hybrids, as well as in large insert cloning vectors. We demonstrate that the canid IRS products are polymorphic and could be developed as genetic markers for filter-based genotyping in this organism. Received: 23 July 1997 / Accepted: 9 September 1997     

Oligonucleotides comprised of alternating 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides and D-2'-deoxyribonucleosides (2'F-ANA/DNA 'altimers') induce efficient RNA cleavage mediated by RNase H

Chimeric oligonucleotides comprised of alternating residues of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (2'F-ANA) and DNA were synthesized and evaluated for an important antisense property-the ability to elicit ribonuclease H (RNase H) degradation of complementary RNA. Experiments used both human RNase HII and Escherichia coli RNase HI. Mixed backbone oligomers comprising alternating three-nucleotide segments of 2'F-ANA and three-nucleotide segments of DNA were the most efficient at eliciting RNase H degradation of target RNA, and were significantly better than oligonucleotides entirely composed of DNA, suggesting that these mixed backbone oligonucleotides may be potent antisense agents.     

Identification of the "sense" -strand of T7 DNA through heteroduplex directed in vitro enzyme synthesis

Summary Enzyme synthesisin vitro directed by T7 heteroduplex DNA is observed when the H strand [the strand assuming the higher density in combination with poly(G,U)], carries the wild allele. This is true for enzyme synthesis mediated byE. coli RNA polymerase or by T7 phage RNA polymerase. Experiments of Wetekam (accompanying paper) using heteroduplexes with markers in thegal operon ofE. coli gave analogous results.     

5'-bis-pyrenylated oligonucleotides displaying excimer fluorescence provide sensitive probes of RNA sequence and structure

Oligonucleotide conjugates bearing two pyrene residues attached to 5′-phosphate through a phosphoramide bond were synthesised. Fluorescence spectra of the conjugates show a peak typical of monomer emission (λ_max 382 nm) and a broad emission peak with λ_max 476 nm, which indicates the excimer formation between the two pyrene residues. Conjugation of these two pyrene residues to the 5′-phosphate of oligonucleotides does not affect the stabilities of heteroduplexes formed by conjugates with the corresponding linear strands. A monomer fluorescence of the conjugates is considerably affected by the heteroduplex formation allowing the conjugates to be used as fluorescent hybridisation probes. The 5′-bis-pyrenylated oligonucleotides have been successfully used for investigation of affinity and kinetics of antisense oligonucleotides binding to the multidrug resistance gene 1 (PGY1/MDR1) mRNA. The changes of excimer fluorescence of the conjugates occurring during hybridisation depended on the structure of the binding sites: hybridisation to heavily structured parts of RNA resulted in quenching of the excimer fluorescence, while binding to RNA regions with a loose secondary structure was accompanied by an enhancement of the excimer fluorescence. Potentially, these conjugates may be considered as fluorescent probes for RNA structure investigation.     

Genetic stability and DNA rearrangements associated with a 2?×?1.1-Kb perfect palindrome in Escherichia coli

We show that circular plasmids containing perfect palindromic regions of 2 × 1.1 kb can be propagated in sbcC strains of Escherichia coli, a result that is at variance with the well known observation that λ DNA cannot tolerate palindromic regions larger than 2 × 265 bp. However, a significant fraction of these palindrome-containing plasmids can be recovered from E. coli strains either as linear molecules with hairpins at their ends or as head-to-head dimers, both in a RuvC-and RusA-independent manner. Our results suggests that large palindromes may form cruciforms in E. coli. However, palindrome-associated DNA rearrangements occur by a process that does not require any known cruciform resolvase activity. Our data support a replication-dependent model for the induction of DNA rearrangements by perfect palindromes. Received: 11 May 1998 / Accepted: 24 June 1998     

Partial purification of an activity from human cells that promotes homologous pairing and the formation of heteroduplex DNA in the presence of ATP

Summary An activity that can promote homologous pairing and strand transfer between suitable DNA substrates has been partially purified from human skin fibroblasts and from Hela cells. The strand transfer reaction was investigated with DNA substrates consisting of single-stranded circular and duplex linear phage DNA. It requires ATP, and under optimal conditions yields heteroduplex molecules containing one strand from each parental DNA substrate. The reactions appears to be of the same general nature as those mediated by RecA proteins of Escherichia coli and the Rec1 protein of Ustilago maydis.     

Promoting DNA molecules association by amphiphilic derivatives of 1,3-diazaadamantanes containing hydrophobic side chains

Earlier, a new class of compounds, amphiphilic derivatives of 1,3-diazaadamantanes, capable of facilitating the strand exchange in the system of short oligonucleotides, has been discovered. Longer hydrophobic side chains in 1,3-diazaadamantanes have been found to promote stronger acceleration of the reaction. In this study, the interaction of two 1,3-diazaadamantane derivatives containing different side chains with DNA was investigated using optical methods. Concentrations of micelle formation by the 1,3-diazaadamantanes, as well as the ranges of concentrations where the compounds/water mixtures exist in the form of true solutions, were determined based on the increase in the fluorescence intensity of 1-anilinonaphthalene-8-sulfonate probe. The affinities of 1,3-diazaadamantanes to DNA were determined with fluorescent intercalator displacement (FID) assay. A significant increase in the hydrodynamic volume of short DNA hairpins in complexes with 1,3-diazaadamantanes was revealed by the estimation of the fluorescence polarization of ethidium bromide probe bound in the hairpins. The intermolecular association of DNA hairpins upon binding with 1,3-diazaadamantanes was confirmed by Förster resonance energy transfer in an equimolar mixture of hairpins fluorescently labeled with Cy-3 or Cy5. In the study, the number of positive charges on 1,3-diazaadamantane derivatives that contain side chains of different lengths was demonstrated to affect their affinity to DNA, while longer hydrophobic side chains ensured more efficient interaction between the DNA duplexes that may facilitate DNA strand exchange.     

Cleavage of double-stranded DNA by manganese tris(methylpyridiniumyl)porphyrin linked to 3′-spermine oligonucleotides

 Cleavage of double-stranded DNA was performed with cationic manganese porphyrin complexes linked via a spermine tether to the 3′- or 5′-side of triple-helix-forming oligonucleotides (cleaver-TFO conjugates). The targeted sequence was a 15-polypurine sequence present in the env gene of HIV-1 (positions 7301–7315). The presently used TFOs contain only thymine and 5-methylcytosine residues and one adenine at the 3′-end in order to be able to easily introduce a 3′-polyamine linker by reductive amination of the corresponding 3′-apurinic polypyrimidine oligonucleotides. With this method we prepared these TFO-cleaver conjugates in 45% yield with only two equivalents of the Mn-TrisMPyP-COOH precursor. These new metalloporphyrin-TFO conjugates were able to cleave a complementary 45-mer duplex at 10 nM concentration with only ten equivalents of TFO-cleaver. Conjugates without spermine, without 5-methylcytosine, with a random sequence or with the managanese porphyrin-spermine entity on the 5′-end of TFOs were synthesized for comparative studies. Received: 6 December 1995 / Accepted: 5 February 1996