DNA Tracts Research Articles

RecX Facilitates Homologous Recombination by Modulating RecA Activities

The Bacillus subtilis recH342 strain, which decreases interspecies recombination without significantly affecting the frequency of transformation with homogamic DNA, carried a point mutation in the putative recX (yfhG) gene, and the mutation was renamed as recX342. We show that RecX (264 residues long), which shares partial identity with the Proteobacterial RecX (<180 residues), is a genuine recombination protein, and its primary function is to modulate the SOS response and to facilitate RecA-mediated recombinational repair and genetic recombination. RecX-YFP formed discrete foci on the nucleoid, which were coincident in time with RecF, in response to DNA damage, and on the poles and/or the nucleoid upon stochastic induction of programmed natural competence. When DNA was damaged, the RecX foci co-localized with RecA threads that persisted for a longer time in the recX context. The absence of RecX severely impaired natural transformation both with plasmid and chromosomal DNA. We show that RecX suppresses the negative effect exerted by RecA during plasmid transformation, prevents RecA mis-sensing of single-stranded DNA tracts, and modulates DNA strand exchange. RecX, by modulating the “length or packing” of a RecA filament, facilitates the initiation of recombination and increases recombination across species.

The role of CSM3, MRC1, and TOF1 in minisatellite stability and large loop DNA repair during meiosis in yeast

Double-stranded break (DSB) repair during meiotic recombination in yeast Saccharomyces cerevisiae leads to the formation of heteroduplex DNA, a hybrid DNA molecule composed of single strands from two homologous chromosomes. Differences in sequence between the strands within heteroduplex DNA generate mismatches or large unpaired loops that are substrates for repair. At least two pathways function to repair large loops that form within heteroduplex DNA: the RAD1-dependent large loop repair (LLR) pathway and another as yet uncharacterized RAD1-independent LLR pathway. Repair of large loops during meiotic recombination is especially important for the genomic stability of the repetitive DNA sequences known as minisatellites. Minisatellite DNA tracts are generally stable during mitotic cell divisions but frequently alter in length during meiosis. Using a yeast minisatellite system in which the human minisatellite associated with the HRAS1 proto-oncogene has been inserted into the recombination hotspot region upstream of HIS4 in S. cerevisiae, our lab previously showed that the RAD1-dependent LLR pathway controls minisatellite length expansions, but not contractions. Here we show that minisatellite length expansions are controlled by the products of the CSM3 and TOF1 genes, while contractions are controlled by MRC1. By examining meiotic segregation patterns in yeast strains heterozygous for the 26bp his4-lopd insert, we found that deleting CSM3 caused a loss of LLR activity similar to that seen in a RAD1 mutant. Double mutant analysis revealed that failure to repair loops is exacerbated upon deleting both RAD1 and CSM3 – specifically the type of repair that fills in loops, which would generate minisatellite length expansions. A model for minisatellite length alteration based on these results is presented.

LINE-1-Derived Poly(A) Microsatellites Undergo Rapid Shortening and Create Somatic and Germline Mosaicism in Mice

Interspersed and tandem repeat sequences comprise the bulk of mammalian genomes. Interspersed repeats result from successive replication by transposable elements, such as Alu and long interspersed element type 1 (L1). Microsatellites are tandem repeats of 1-6 base pairs, among which poly(A) microsatellites are the most abundant in the human genome. The rise and fall of a microsatellite has been depicted as a life cycle. Previous studies have demonstrated that Alu and L1 insertions are a major source of A-rich microsatellites owing to the concurrent formation of a poly(A) DNA tract at the 3'-end of each insertion. The fate of such poly(A) tracts has been studied by surveying the length distribution of genomic resident Alu and L1 insertions. However, these cross-sectional studies provide no information about the tempo of mutation immediately after birth. In this study, de novo L1 insertions were created using a transgenic L1 mouse model and traced through generations to investigate the early life of poly(A) microsatellites. High frequencies of intra-individual and intergenerational shortening were observed for long poly(A) tracts, creating somatic and germline mosaicism at the insertion site, whereas little variation was observed for short poly(A) alleles. As poly(A) microsatellites are the major intrinsic signal for nucleosome positioning, their remarkable abundance and variability make them a significant source of epigenetic variation. Thus, the birth of poly(A) microsatellites from retrotransposons and the subsequent rapid and variable shortening represent a new way with which retrotransposons can modify the genetic and epigenetic architecture of our genome.

A triple-helix forming oligonucleotide targeting genomic DNA fails to induce mutation

Purine tracts in duplex DNA can bind oligonucleotide strands in a sequence specific manner to form triple-helix structures. Triple-helix forming oligonucleotides (TFOs) targeting supFG1 constructs have previously been shown to be mutagenic raising safety concerns for oligonucleotide-based pharmaceuticals. We have engineered a TFO, TFO27, to target the genomic Hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus to define the mutagenic potential of such structures at genomic DNA. We report that TFO27 was resistant to nuclease degradation and readily binds to its target motif in a cell free system. Contrary to previous studies using the supFG1 reporter construct, TFO27 failed to induce mutation within the genomic HPRT locus. We suggest that it is possible that previous reports of triplex-mediated mutation using the supFG1 reporter construct could be confounded by DNA quadruplex formation. Although the present study indicates that a TFO targeting a genomic locus lacks mutagenic activity, it is unclear if this finding can be generalised to all TFOs and their targets. For the present, we suggest that it is prudent to avoid large purine stretches in oligonucleotide pharmaceutical design to minimise concern regarding off-target genotoxicity.

Telomeric DNA damage is irreparable and causes persistent DNA-damage-response activation

The DNA-damage response (DDR) arrests cell-cycle progression until damage is removed. DNA-damage-induced cellular senescence is associated with persistent DDR. The molecular bases that distinguish transient from persistent DDR are unknown. Here we show that a large fraction of exogenously induced persistent DDR markers is associated with telomeric DNA in cultured cells and mammalian tissues. In yeast, a chromosomal DNA double-strand break next to a telomeric sequence resists repair and impairs DNA ligase 4 recruitment. In mammalian cells, ectopic localization of telomeric factor TRF2 next to a double-strand break induces persistent DNA damage and DDR. Linear, but not circular, telomeric DNA or scrambled DNA induces a prolonged checkpoint in normal cells. In terminally differentiated tissues of old primates, DDR markers accumulate at telomeres that are not critically short. We propose that linear genomes are not uniformly reparable and that telomeric DNA tracts, if damaged, are irreparable and trigger persistent DDR and cellular senescence.

Label-free, atomic force microscopy-based mapping of DNA intrinsic curvature for the nanoscale comparative analysis of bent duplexes

We propose a method for the characterization of the local intrinsic curvature of adsorbed DNA molecules. It relies on a novel statistical chain descriptor, namely the ensemble averaged product of curvatures for two nanosized segments, symmetrically placed on the contour of atomic force microscopy imaged chains. We demonstrate by theoretical arguments and experimental investigation of representative samples that the fine mapping of the average product along the molecular backbone generates a characteristic pattern of variation that effectively highlights all pairs of DNA tracts with large intrinsic curvature. The centrosymmetric character of the chain descriptor enables targetting strands with unknown orientation. This overcomes a remarkable limitation of the current experimental strategies that estimate curvature maps solely from the trajectories of end-labeled molecules or palindromes. As a consequence our approach paves the way for a reliable, unbiased, label-free comparative analysis of bent duplexes, aimed to detect local conformational changes of physical or biological relevance in large sample numbers. Notably, such an assay is virtually inaccessible to the automated intrinsic curvature computation algorithms proposed so far. We foresee several challenging applications, including the validation of DNA adsorption and bending models by experiments and the discrimination of specimens for genetic screening purposes.

Mutation Hot Spots in Yeast Caused by Long-Range Clustering of Homopolymeric Sequences

Evolutionary theory assumes that mutations occur randomly in the genome; however, studies performed in a variety of organisms indicate the existence ofcontext-dependent mutation biases. Sources ofmutagenesis variation across large genomic contexts (e.g., hundreds of bases) have not been identified. Here, we use high-coverage whole-genome sequencing of a conditional mismatch repair mutant line of diploid yeast to identify mutations thataccumulated after 160 generations of growth. The vast majority of the mutations accumulated asinsertion/deletions (in/dels) in homopolymeric [poly(dA:dT)] and repetitive DNA tracts. Surprisingly, the likelihood of an in/del mutation in a given poly(dA:dT) tract is increased by the presence of nearby poly(dA:dT) tracts in up to a 1,000bp region centered on the given tract. Our work suggests that specific mutation hot spots can contribute disproportionately to the genetic variation that is introduced into populations and provides long-range genomic sequence context that contributes to mutagenesis.

Evidence for ATP-dependent Structural Rearrangement of Nuclease Catalytic Site in DNA Mismatch Repair Endonuclease MutL

DNA mismatch repair (MMR) greatly contributes to genome integrity via the correction of mismatched bases that are mainly generated by replication errors. Postreplicative MMR excises a relatively long tract of error-containing single-stranded DNA. MutL is a widely conserved nicking endonuclease that directs the excision reaction to the error-containing strand of the duplex by specifically nicking the daughter strand. Because MutL apparently exhibits nonspecific nicking endonuclease activity in vitro, the regulatory mechanism of MutL has been argued. Recent studies suggest ATP-dependent conformational and functional changes of MutL, indicating that the regulatory mechanism involves the ATP binding and hydrolysis cycle. In this study, we investigated the effect of ATP binding on the structure of MutL. First, a cross-linking experiment confirmed that the N-terminal ATPase domain physically interacts with the C-terminal endonuclease domain. Next, hydrogen/deuterium exchange mass spectrometry clarified that the binding of ATP to the N-terminal domain induces local structural changes at the catalytic sites of MutL C-terminal domain. Finally, on the basis of the results of the hydrogen/deuterium exchange experiment, we successfully identified novel regions essential for the endonuclease activity of MutL. The results clearly show that ATP modulates the nicking endonuclease activity of MutL via structural rearrangements of the catalytic site. In addition, several Lynch syndrome-related mutations in human MutL homolog are located in the position corresponding to the newly identified catalytic region. Our data contribute toward understanding the relationship between mutations in MutL homolog and human disease.

Schizosaccharomyces pombe Protection of Telomeres 1 Utilizes Alternate Binding Modes To Accommodate Different Telomeric Sequences

The ends of eukaryotic chromosomes consist of long tracts of repetitive GT-rich DNA with variable sequence homogeneity between and within organisms. Telomeres terminate in a conserved 3'-ssDNA overhang that, regardless of sequence variability, is specifically and tightly bound by proteins of the telomere-end protection family. The high affinity ssDNA-binding activity of S. pombe Pot1 protein (SpPot1) is conferred by a DNA-binding domain consisting of two subdomains, Pot1pN and Pot1pC. Previous work has shown that Pot1pN binds a single repeat of the core telomere sequence (GGTTAC) with exquisite specificity, while Pot1pC binds an extended sequence of nine nucleotides (GGTTACGGT) with modest specificity requirements. We find that full-length SpPot1 binds the composite 15mer, (GGTTAC)(2)GGT, and a shorter two-repeat 12mer, (GGTTAC)(2), with equally high affinity (<3 pM), but with substantially different kinetic and thermodynamic properties. The binding mode of the SpPot1/15mer complex is more stable than that of the 12mer complex, with a 2-fold longer half-life and increased tolerance to nucleotide and amino acid substitutions. Our data suggest that SpPot1 protection of heterogeneous telomeres is mediated through 5'-sequence recognition and the use of alternate binding modes to maintain high affinity interaction with the G-strand, while simultaneously discriminating against the complementary strand.

No evidence of substantia nigra telomere shortening in Parkinson's disease

Telomeres are repetitive tracts of DNA which protect chromosomal integrity. Increased oxidative stress leads to shorter telomeres, which have been associated with several late-onset human diseases. Given independent evidence of oxidative stress and Parkinson's disease (PD), and conflicting reports of the role of telomere length in PD, we measured telomere length in both PD peripheral blood monocytes and in substantia nigra from affected individuals and controls. We confirmed previous findings of a paradoxically longer telomere length in blood from PD patients, but found no difference in telomere length in substantia nigra. Confounding factors provide a likely explanation for the findings in blood, and possibly the reduced frequency of cigarette smoking in PD patients. We conclude that telomere shortening is unlikely to be involved in the pathogenesis of PD.

Microsatellite instability in the smoldering form of adult T-cell leukemia/lymphoma (ATL) in Brazil

Background Microsatellites are short, repetitive elements in genomic DNA that are nonrandomly distributed throughout the human genome. Microsatellite instability (MSI) originates from the systematic accumulation of uncorrected deletion/insertion in repetitive DNA tracts in cancer cells with a deficient mismatch repair system. In ATL, the presence of MSI has been shown in aggressive types, principally in acute ATL. However, few studies have evaluated MSI in smoldering ATL. The aim of this study was to investigate MSI in patients from Bahia (Brazil) with ATL, including the smoldering subtypes.

Genome-Wide Association Study of Relative Telomere Length

Telomere function is essential to maintaining the physical integrity of linear chromosomes and healthy human aging. The probability of forming proper telomere structures depends on the length of the telomeric DNA tract. We attempted to identify common genetic variants associated with log relative telomere length using genome-wide genotyping data on 3,554 individuals from the Nurses' Health Study and the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial that took part in the National Cancer Institute Cancer Genetic Markers of Susceptibility initiative for breast and prostate cancer. After genotyping 64 independent SNPs selected for replication in additional Nurses' Health Study and Women's Genome Health Study participants, we did not identify genome-wide significant loci; however, we replicated the inverse association of log relative telomere length with the minor allele variant [C] of rs16847897 at the TERC locus (per allele β = −0.03, P = 0.003) identified by a previous genome-wide association study. We did not find evidence for an association with variants at the OBFC1 locus or other loci reported to be associated with telomere length. With this sample size we had >80% power to detect β estimates as small as ±0.10 for SNPs with minor allele frequencies of ≥0.15 at genome-wide significance. However, power is greatly reduced for β estimates smaller than ±0.10, such as those for variants at the TERC locus. In general, common genetic variants associated with telomere length homeostasis have been difficult to detect. Potential biological and technical issues are discussed.

Multiple POT1–TPP1 Proteins Coat and Compact Long Telomeric Single-Stranded DNA

Telomeres are nucleoprotein complexes that cap and protect the ends of linear chromosomes. In humans, telomeres end in 50–300 nt of G-rich single-stranded DNA (ssDNA) overhangs. Protection of telomeres 1 (POT1) binds with nanomolar affinity to the ssDNA overhangs and forms a dimer with another telomere-end binding protein called TPP1. Whereas most previous studies utilized telomeric oligonucleotides comprising single POT1–TPP1 binding sites, here, we examined 72- to 144-nt tracts of telomeric DNA containing 6–12 POT1–TPP1 binding sites. Using electrophoretic mobility gel shift assays, size-exclusion chromatography, and electron microscopy, we analyzed telomeric nucleoprotein complexes containing POT1 alone, POT1–TPP1, and a truncated version of POT1 (POT1-N) that maintains its DNA-binding domain. The results revealed that POT1-N and POT1–TPP1 can completely coat long telomeric ssDNA substrates. Furthermore, we show that ssDNA coated with human POT1–TPP1 heterodimers forms compact, potentially ordered structures.

Poly(T) Variation in Heteroderid Nematode Mitochondrial Genomes is Predominantly an Artefact of Amplification

We assessed the rate of in vitro polymerase errors at polythymidine [poly(T)] tracts in the mitochondrial DNA (mtDNA) of a heteroderid nematode (Heterodera cajani). The mtDNA of these nematodes contain unusually high numbers of poly(T) tracts, and have previously been suggested to contain biological poly(T) length variation. However, using a cloned molecule, we observed that poly(T) variation was generated in vitro at regions containing more than six consecutive Ts. This artefactual error rate was estimated at 7.3 × 10(-5) indels/poly(T) tract >6 Ts/cycle. This rate was then compared to the rate of poly(T) variation detected after the amplification of a biological sample, in order to estimate the 'biological + artefactual' rate of poly(T) variation. There was no significant difference between the artefactual and the artefactual + biological rates, suggesting that the majority of poly(T) variation in the biological sample was artefactual. We then examined the generation of poly(T) variation in a range of templates with tracts up to 16 Ts long, utilizing a range of Heteroderidae species. We observed that T deletions occurred five times more frequently than insertions, and a trend towards increasing error rates with increasing poly(T) tract length. These findings have significant implications for studies involving genomes with many homopolymer tracts.

Direct and indirect effects of H-NS and Fis on global gene expression control in Escherichia coli

Nucleoid-associated proteins (NAPs) are global regulators of gene expression in Escherichia coli, which affect DNA conformation by bending, wrapping and bridging the DNA. Two of these—H-NS and Fis—bind to specific DNA sequences and structures. Because of their importance to global gene expression, the binding of these NAPs to the DNA was previously investigated on a genome-wide scale using ChIP-chip. However, variation in their binding profiles across the growth phase and the genome-scale nature of their impact on gene expression remain poorly understood. Here, we present a genome-scale investigation of H-NS and Fis binding to the E. coli chromosome using chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq). By performing our experiments under multiple time-points during growth in rich media, we show that the binding regions of the two proteins are mutually exclusive under our experimental conditions. H-NS binds to significantly longer tracts of DNA than Fis, consistent with the linear spread of H-NS binding from high- to surrounding lower-affinity sites; the length of binding regions is associated with the degree of transcriptional repression imposed by H-NS. For Fis, a majority of binding events do not lead to differential expression of the proximal gene; however, it has a significant indirect effect on gene expression partly through its effects on the expression of other transcription factors. We propose that direct transcriptional regulation by Fis is associated with the interaction of tandem arrays of Fis molecules to the DNA and possible DNA bending, particularly at operon-upstream regions. Our study serves as a proof-of-principle for the use of ChIP-seq for global DNA-binding proteins in bacteria, which should become significantly more economical and feasible with the development of multiplexing techniques.

Prediction of Nucleosome Positioning in Genomes: Limits and Perspectives of Physical and Bioinformatic Approaches

Nucleosomes, the fundamental repeating subunits of all eukaryotic chromatin, are responsible for packaging DNA into chromosomes inside the cell nucleus and controlling gene expression. While it has been well established that nucleosomes exhibit higher affinity for select DNA sequences, until recently it was unclear whether such preferences exerted a significant, genome-wide effect on nucleosome positioning in vivo. For this reason, an increasing interest is arising on a wide-ranging series of experimental and computational analyses capable of predicting the nucleosome positioning along genomes. Toward this goal, we propose a theoretical model for predicting nucleosome thermodynamic stability in terms of DNA sequence. Based on a statistical mechanical approach, the model allows the calculation of the sequence-dependent canonical ensemble free energy involved in nucleosome formation. The theoretical free energies were evaluated for 90 single nucleosome DNA tracts and successfully compared with those obtained with nucleosome competitive reconstitution. These results, obtained for single nucleosomes, could in principle allow the calculation of the intrinsic affinity of nucleosomes along DNA sequences virtually opening the possibility of predicting the nucleosome positioning along genomes on physical basis. The theoretical nucleosome distribution was compared and validated with that of yeast and human genome experimentally determined. The results interpret on a physical basis the experimental nucleosome positioning and are comparable with those obtained adopting models based on the identification of some recurrent sequence features obtained from the statistical analysis of a very large pool of nucleosomal DNA sequences provided by the positioning maps of genomes.

Slip into Something More Functional: Selection Maintains Ancient Frameshifts in Homopolymeric Sequences

Mutational hotspots offer significant sources of genetic variability upon which selection can act. However, with a few notable exceptions, we know little about the dynamics and fitness consequences of mutations in these regions. Here, we explore evolutionary forces shaping homopolymeric tracts that are especially vulnerable to slippage errors during replication and transcription. Such tracts are typically eliminated by selection from most bacterial sequences, yet persist in genomes of endosymbionts with small effective population sizes (N(e)) and biased base compositions. Focusing on Blochmannia, a bacterial endosymbiont of ants, we track the divergence of genes that contain frameshift mutations within long (9-11 bp) polyA or polyT tracts. Earlier experimental work documented that transcriptional slippage restores the reading frame in a fraction of messenger RNA molecules and thereby rescues the function of frameshifted genes. In this study, we demonstrate a surprising persistence of these frameshifts and associated tracts for millions of years. Across the genome of this ant mutualist, rates of indel mutation within homopolymeric tracts far exceed the synonymous mutation rate, indicating that long-term conservation of frameshifts within these tracts is inconsistent with neutrality. In addition, the homopolymeric tracts themselves are more conserved than expected by chance, given extensive neutral substitutions that occur elsewhere in the genes sampled. These data suggest an unexpected role for slippage-prone DNA tracts and highlight a new mechanism for their persistence. That is, when such tracts contain a frameshift, transcriptional slippage plays a critical role in rescuing gene function. In such cases, selection will purge nucleotide changes interrupting the slippery tract so that otherwise volatile sequences become frozen in evolutionary time. Although the advantage of the frameshift itself is less clear, it may offer a mechanism to lower effective gene expression by reducing but not eliminating transcripts that encode full-length proteins.

Transplatin-Conjugated Triplex-Forming Oligonucleotides Form Adducts with Both Strands of DNA

Triplex-forming oligonucleotides (TFOs) can bind to polypurine x polypyrimidine tracts in DNA and, as a consequence, perturb the normal functioning of a targeted gene. The effectiveness of such antigene TFOs can potentially be enhanced by covalent attachment of the TFO to its DNA target. Here, we report that attachment of N-7-platinated guanine nucleosides to the 3'- and/or 5'-ends of oligopyrimidine TFOs enables these TFOs to form highly stable adducts with target DNA deoxyguanosines or deoxyadenosines that are adjacent to the TFO binding site. Such adduct formation stably anchors the TFO to its target. Depending on the sequences adjacent to the TFO binding site, adduct formation can occur on either strand of the DNA. Adduct formation by 3',5'-bis-platinated TFOs can result in the formation of an interstrand cross-link between both strands of the DNA duplex. Formation of the adducts, which could be reversed by treatment with sodium cyanide, was dependent upon the ability of the TFO to bind to DNA and appeared to occur at a rate slower than that at which the TFO bound to the DNA duplex. The extent of adduct formation at 37 degrees C by platinated deoxyribo-TFOs diminished as the pH was increased from 6.5 to 7.4. In contrast, high levels (approximately 86%) of adduct formation by platinated 2'-O-methylribo-TFOs were observed at both pH 6.5 and pH 7.4. Platinated 2'-O-methylribo-TFOs were also shown to bind to plasmid DNA and inhibit transcription in vitro, and to inhibit plasmid replication in E. coli cells. These results suggest that platinum-conjugated TFOs may be good candidates for use as antigene agents.

Intrinsic hTRF1 fluorescence quenching reveals details of telomere DNA binding activity: Impact of DNA length, structure and position of telomeric repeats

The myb-DNA binding domain is characterized by a 3-alpha helical bundle and three repeats of this domain drive sequence specific DNA binding of the c-myb transcription factor. Human TRF1 contains a single myb-related domain and as a homodimer, enables the sequence specific binding of telomeric DNA. In this report we provide a kinetic assessment of hTRF1 DNA binding activity. Using intrinsic fluorescence quenching we present evidence that hTRF1 binds to both telomeric and non-telomeric DNA with kinetic discrimination to allow stable binding to telomeric tracts of DNA. The position of telomere repeats does not impact binding though the number of repeats and structure does impact binding. Kinetic analysis of DNA-dependent intrinsic tryptophan fluorescence quenching of hTRF1 revealed a two step binding process that is impacted by telomere repeat length, position, and structure. These data are consistent with existing structural and equilibrium binding data for hTRF1 recognition and binding of telomere DNA.

Optimization of the sequence of twisted intercalating nucleic acids (TINA) forming triple helix with the polypurine tract of the proviral HIV DNA

Twisted intercalating nucleic acids form stable triplexes with polypurine tracts of double-stranded DNA. Their affinity depends on their length, primary structure and base contents, parallel or antiparallel orientation of oligonucleotides respectively to DNA, number of TINA residues and their relative positions. Basing on parallel CT, GT and antiparallel GT triplex-forming 16-mer oligonucleotides targeted to polypurine tract of HIV proviral DNA, we synthesized eleven different oligonucleotides with 2-4 TINA insertions in different positions. Studies of their interaction with target duplex by gel shift, fluorescence spectroscopy, circular dichroism and thermal denaturation demonstrated that antiparallel GT oligonucleotides form more stable triplexes than parallel TC or TG ones. Two best candidates were selected for the further studies. The first one (5'-AGGGxGGGTTTxTGTTTT-3', Kd = 219 nM) contains only two TINA insertions and does not aggregate in non-denaturing conditions, in contrast to majority of other oligonucleotides.