Single-stranded Loop Regions Research Articles

Mutational impact of APOBEC3A and APOBEC3B in a human cell line and comparisons to breast cancer.

A prominent source of mutation in cancer is single-stranded DNA cytosine deamination by cellular APOBEC3 enzymes, which results in signature C-to-T and C-to-G mutations in TCA and TCT motifs. Although multiple enzymes have been implicated, reports conflict and it is unclear which protein(s) are responsible. Here we report the development of a selectable system to quantify genome mutation and demonstrate its utility by comparing the mutagenic activities of three leading candidates-APOBEC3A, APOBEC3B, and APOBEC3H. The human cell line, HAP1, is engineered to express the thymidine kinase (TK) gene of HSV-1, which confers sensitivity to ganciclovir. Expression of APOBEC3A and APOBEC3B, but not catalytic mutant controls or APOBEC3H, triggers increased frequencies of TK mutation and similar TC-biased cytosine mutation profiles in the selectable TK reporter gene. Whole genome sequences from independent clones enabled an analysis of thousands of single base substitution mutations and extraction of local sequence preferences with APOBEC3A preferring YTCW motifs 70% of the time and APOBEC3B 50% of the time (Y = C/T; W = A/T). Signature comparisons with breast tumor whole genome sequences indicate that most malignancies manifest intermediate percentages of APOBEC3 signature mutations in YTCW motifs, mostly between 50 and 70%, suggesting that both enzymes contribute in a combinatorial manner to the overall mutation landscape. Although the vast majority of APOBEC3A- and APOBEC3B-induced single base substitution mutations occur outside of predicted chromosomal DNA hairpin structures, whole genome sequence analyses and supporting biochemical studies also indicate that both enzymes are capable of deaminating the single-stranded loop regions of DNA hairpins at elevated rates. These studies combine to help resolve a long-standing etiologic debate on the source of APOBEC3 signature mutations in cancer and indicate that future diagnostic and therapeutic efforts should focus on both APOBEC3A and APOBEC3B.

Abstract P5-12-01: Apobec mutation signature in breast cancer explained by combinatorial action of apobec3a and apobec3b

Abstract Background: Mutations drive the initiation and progression of cancer. A leading druggable source of mutation in cancer is enzymatic deamination of single-stranded DNA cytosines by cellular APOBEC3 enzymes. Cytosine-to-uracil deamination can result in a variety of different mutational outcomes including DNA breakage and chromosomal aberrations as well as single base substitution mutations. The latter are comprised of C-to-T and C-to-G mutations in TCA or TCT trinucleotides and attributable to the intrinsic preference of several APOBEC3 family members for binding to these motifs. This mutation pattern, commonly called the “APOBEC signature”, is evident in approximately one-quarter of primary breast tumors and one-third of metastatic breast tumors. Although multiple APOBEC3 enzymes have been implicated as a source of this signature in breast cancer (namely, APOBEC3A, APOBEC3B, and APOBEC3H), the literature is full of conflicting views and it is not clear which of these enzymes contributes most significantly to the mutational landscape of breast cancer. Methods: The near-haploid human cell line, HAP1, was engineered to express the HSV-1 TK gene as a mutation reporter. Candidate APOBEC3 enzymes were expressed individually and confirmed by immunoblotting and activity assays. DNA breakage was measured directly by COMET assays and DNA damage responses indirectly by phosphorylated gamma-H2AX staining. Mutation frequencies were quantified by assaying rates of drug resistance, and mutation patterns were analyzed by sequencing locally in TK and globally across whole genomes. Results: APOBEC3A and APOBEC3B both caused significant increases in chromosomal DNA breakage and DNA damage responses. These enzymes also elevated drug resistance mutation frequencies. In contrast, expression of active APOBEC3H or catalytic mutant derivatives of APOBEC3A and APOBEC3B failed to trigger increases beyond normal spontaneous levels. Interestingly, APOBEC3A and APOBEC3B both inflicted mutation signatures that were indistinguishable locally in TK and globally across whole genomes. The vast majority of these APOBEC signature mutations were dispersed (non-kataegic) and not associated with obvious mesoscale chromosomal features such as single-stranded loop regions of stem-loop structures. Computational comparisons of the broader pentanucleotide APOBEC3A and APOBEC3B mutation signatures and those extracted from 794 primary breast tumor genomes (ICGC cohort) revealed an APOBEC3A-biased subset, an APOBEC3B-biased subset, and a larger group of tumors best explained by combinatorial action of both of these enzymes. Conclusions: Our results indicate that APOBEC3A and APOBEC3B contribute combinatorially in most instances to the observed APOBEC mutation signature in breast cancer. These results provide a framework for developing diagnostic and therapeutic approaches for APOBEC-positive breast cancer. Citation Format: Reuben S Harris, Matthew C Jarvis, Michael A Carpenter, Margaret R Brown, Prokopios P Argyris, William Brown, Douglas Yee. Apobec mutation signature in breast cancer explained by combinatorial action of apobec3a and apobec3b [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-01.

Non-crosslinking gold nanoprobe-LAMP for simple, colorimetric, and specific detection of Salmonella typhi

In this study, we developed a non-crosslinking gold nanoprobe loop-mediated isothermal amplification (LAMP) method for nanodiagnosis of bacterial typhoid fever source, Salmonella typhi. Therefore, a unique region in the S. typhi genomic DNA was targeted for LAMP amplification using a specific set of four precisely designed primers. Also, for specific colorimetric visualization of the amplicons, a thiolated oligonucleotide probe, complementary to the single-stranded loop region of the amplicons between F2 and F1C segments, was designed. The probe was bound to the surface of gold nanoparticles via covalent bonds. Increasing the salt concentration in the detection reaction medium led to aggregation of nanoprobes in the blank and the negative vessels in a time-dependent form. That was followed by a change in the surface plasmon resonance (SPR) leading to blue/black color that was observable by the naked eyes after about 5 min. Meanwhile, the original pink/red color was retained in the positive sample due to the large interparticle spaces and the stability against the ionic strength elevation which persisted for about 30 min. The whole process of DNA extraction, amplification, and detection took less than 2 h with a sensitivity of 20 CFU/ml. The developed gold nanoprobe-LAMP could serve as a simple, rapid, and cost-effective method for nanodiagnosis of S. typhi in point-of-need applications.

An introduction to recurrent nucleotide interactions in RNA.

RNA secondary structure diagrams familiar to molecular biologists summarize at a glance the folding of RNA chains to form Watson–Crick paired double helices. However, they can be misleading: First of all, they imply that the nucleotides in loops and linker segments, which can amount to 35% to 50% of a structured RNA, do not significantly interact with other nucleotides. Secondly, they give the impression that RNA molecules are loosely organized in three-dimensional (3D) space. In fact, structured RNAs are compactly folded as a result of numerous long-range, sequence-specific interactions, many of which involve loop or linker nucleotides. Here, we provide an introduction for students and researchers of RNA on the types, prevalence, and sequence variations of inter-nucleotide interactions that structure and stabilize RNA 3D motifs and architectures, using Escherichia coli (E. coli) 16S ribosomal RNA as a concrete example. The picture that emerges is that almost all nucleotides in structured RNA molecules, including those in nominally single-stranded loop or linker regions, form specific interactions that stabilize functional structures or mediate interactions with other molecules. The small number of noninteracting, ‘looped-out’ nucleotides make it possible for the RNA chain to form sharp turns. Base-pairing is the most specific interaction in RNA as it involves edge-to-edge hydrogen bonding (H-bonding) of the bases. Non-Watson–Crick base pairs are a significant fraction (30% or more) of base pairs in structured RNAs.

Bidirectional processing of pri-miRNAs with branched terminal loops by Arabidopsis Dicer-like1

miRNAs originate from primary transcripts (pri-miRNAs) with characteristic stem-loop structures. Accurate processing of pri-miRNAs is required for functional miRNAs. Here, using pri-miR166 family as a paradigm, we report the decisive role of pri-miRNA terminal loops in miRNA biogenesis. We found that multi-branched terminal loops in pri-miR166s substantially suppressed miR166 expression in vivo. Unlike canonical processing of pri-miRNAs, terminal-loop-branched (TLBed) pri-miRNAs can be processed by Dicer-like1 (DCL1) complexes bi-directionally: from base to loop and from loop to base, resulting in productive and abortive processing of miRNAs, respectively. In either case, DCL1 complexes canonically cut pri-miRNAs at a distance of 16-17 base pairs (bp) from a reference single-stranded loop region. DCL1 also adjusts processing sites toward an internal loop through its helicase domain. Thus, these results provide new insight into the poorly understood processing mechanism of pri-miRNAs with complicated secondary structures.

Loop-miRs: active microRNAs generated from single-stranded loop regions

MicroRNAs (miRNAs) are key mediators of post-transcriptional gene regulation. The miRNA precursors are processed by the endonucleases Drosha and Dicer into a duplex, bound to an Argonaute protein and unwound into two single-stranded miRNAs. Although alternative ways to generate miRNAs have been discovered, e.g. pre-miRNA cleavage by Ago2 or cleavage products of snoRNAs or tRNAs, all known pathways converge on a double-stranded RNA duplex. Exogenous single-stranded siRNAs (ss-siRNAs) can elicit an effective RNA interference reaction; recent studies have identified chemical modifications increasing their stability and activity. Here, we provide first evidence that endogenous, unmodified, single-stranded RNA sequences are generated from single-stranded loop regions of human pre-miRNA hairpins, the so called loop-miRs. Luciferase assays and immunoprecipitation validate loop-miR activity and incorporation into RNA-induced silencing complexes. This study identifies endogenous miRNAs that are generated from single-stranded regions; hence, it provides evidence that precursor-miRNAs can give rise to three distinct endogenous miRNAs: the guide strand, the passenger strand and the loop-miR.

Pharmacological Characterization of Chemically Synthesized Monomeric phi29 pRNA Nanoparticles for Systemic Delivery

Previous studies have shown that the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor folds into a compact structure, constituting a RNA nanoparticle that can be modularized with functional groups as a nanodelivery system. pRNA nanoparticles can also be self-assembled by the bipartite approach without altering folding property. The present study demonstrated that 2'-F-modified pRNA nanoparticles were readily manufactured through this scalable bipartite strategy, featuring total chemical synthesis and permitting diverse functional modularizations. The RNA nanoparticles were chemically and metabolically stable and demonstrated a favorable pharmacokinetic (PK) profile in mice (half-life (T(1/2)): 5-10 hours, clearance (Cl): <0.13 l/kg/hour, volume of distribution (V(d)): 1.2 l/kg). It did not induce an interferon (IFN) response nor did it induce cytokine production in mice. Repeat intravenous administrations in mice up to 30 mg/kg did not result in any toxicity. Fluorescent folate-pRNA nanoparticles efficiently and specifically bound and internalized to folate receptor (FR)-bearing cancer cells in vitro. It also specifically and dose-dependently targeted to FR(+) xenograft tumor in mice with minimal accumulation in normal tissues. This first comprehensive pharmacological study suggests that the pRNA nanoparticle had all the preferred pharmacological features to serve as an efficient nanodelivery platform for broad medical applications.

Amplified Surface Plasmon Resonance Based DNA Biosensors, Aptasensors, and Hg2+ Sensors Using Hemin/G‐Quadruplexes and Au Nanoparticles

Thiolated nucleic acid hairpin nanostructures that include in their stem region a "caged" G-quadruplex sequence, and in their single-stranded loop region oligonucleotide recognition sequences for DNA, adenosine monophosphate (AMP), or Hg(2+) ions were linked to bare Au surfaces or to Au nanoparticles (NPs) linked to Au surfaces. The opening of the hairpin nanostructures associated with the bare Au surface by the complementary target DNA, AMP substrate, or Hg(2+) ions, in the presence of hemin, led to the self-assembly of hemin/G-quadruplexes on the surface. The resulting dielectric changes on the surface exhibited shifts in the surface plasmon resonance (SPR) spectra, thus providing a readout signal for the recognition events. A similar opening of the hairpin nanostructures, immobilized on the Au NPs associated with the Au surface, by the DNA, AMP, or Hg(2+) led to an ultrasensitive SPR-amplified detection of the respective analytes. The amplification originated from the coupling between the localized surface plasmon associated with the NPs and the surface plasmon wave, an effect that cooperatively amplifies the SPR shifts that result from the formation of the hemin/G-quadruplexes. The different sensing platforms reveal impressive sensitivities and selectivities toward the target analytes.

Mitochondrial RNA Polymerase Is Needed for Activation of the Origin of Light-Strand DNA Replication

Mitochondrial DNA is replicated by a unique enzymatic machinery, which is distinct from the replication apparatus used for copying the nuclear genome. We examine here the mechanisms of origin-specific initiation of lagging-strand DNA synthesis in human mitochondria. We demonstrate that the mitochondrial RNA polymerase (POLRMT) is the primase required for initiation of DNA synthesis from the light-strand origin of DNA replication (OriL). Using only purified POLRMT and DNA replication factors, we can faithfully reconstitute OriL-dependent initiation in vitro. Leading-strand DNA synthesis is initiated from the heavy-strand origin of DNA replication and passes OriL. The single-stranded OriL is exposed and adopts a stem-loop structure. At this stage, POLRMT initiates primer synthesis from a poly-dT stretch in the single-stranded loop region. After about 25 nt, POLRMT is replaced by DNA polymerase gamma, and DNA synthesis commences. Our findings demonstrate that POLRMT can function as an origin-specific primase in mammalian mitochondria.

Secondary structural analysis of the mRNA regions encoding the hemagglutinin cleavage site basic amino acids of the avian influenza virus H5N1 subtype samples

Here we report the codon bias and the mRNA secondary structural features of the hemagglutinin (HA) cleavage site basic amino acid regions of avian influenza virus H5N1 subtypes. We have developed a dynamic extended folding strategy to predict RNA secondary structure with RNAstructure 4.1 program in an iterative extension process. Statistical analysis of the sequences showed that the HA cleavage site basic amino acids favor the adenine-rich codons, and the corresponding mRNA fragments are mainly in the folding states of single-stranded loops. Our sequential and structural analyses showed that to prevent and control these highly pathogenic viruses, that is, to inhibit the gene expression of avian influenza virus H5N1 subtypes, we should consider the single-stranded loop regions of the HA cleavage site-coding sequences as the targets of RNA interference.

RNA Interference Induced by Transient or Stable Expression of Hairpin Structures of Double-Stranded RNA in Drosophila and Mammalian Cells

RNA interference (RNAi) may be induced by a plasmid with an inverted repeat (IR) sequence directing transcription of hairpin-type double-stranded RNA (dsRNA). This study examines the effects of changing various parameters of IR constructs on Drosophila and mammalian RNAi, using the dual luciferase system. RNAi activity was found to vary depending on IR length as well as the length and sequence of the internal loop separating sense and antisense sequences. Both transient and stable RNAi occurred in Drosophila cultured cells. Although transient DNA-mediated RNAi was noted in most mammalian cells, no mammalian cells stably possessing IR sequences and hence RNAi activity could be obtained. In Drosophila, DNA-mediated RNAi was considerably weaker than long-dsRNA-mediated RNAi. The cytological data indicated that this was most probably caused by abortive processing of hairpin RNA produced within cells. DNA-mediated RNAi was examined at the level of Drosophila individuals using extramacrochaetae as a model gene, and the presence of an intron sequence in the single-stranded loop region was shown to be essential for effective RNAi.

Gene silencing in mammalian cells by preformed small RNA duplexes

Small interfering RNAs (siRNAs) mediate RNA interference (RNAi), a process in which target mRNAs are degraded. Here, we have investigated the efficacy of preformed siRNAs to modulate the expression of protein kinase Cα (PKCα) and green fluorescent protein (GFP) in mammalian cells. We show that specific inhibition of PKCα and GFP can be achieved by in vitro transcribed siRNAs. Interestingly, a transcript harboring two self-complementary siRNAs interrupted by a single-stranded loop region inhibited both PKCα and GFP gene expression. These results suggest that the long transcript is processed by single-stranded ribonucleases and/or other proteins into two functional siRNAs. Incubation of the in vitro transcribed bispecific siRNA with protein extracts from HEK 293T cells yielded RNA duplexes similar to the synthetic single siRNA. Taken together, the present data indicate that in vitro transcribed siRNA can be useful for silencing gene expression. Additionally, bi- and perhaps poly-siRNAs may be expressed and processed in mammalian cells.

Ecdysone-controlled mRNA stability in Drosophila salivary glands: deadenylation-independent degradation of larval glue protein gene message during the larval/prepupal transition

20-Hydroxyecdysone induces poly(A) shortening and the subsequent degradation of transcripts encoding the larval glue protein LGP-1 in Drosophila virilis late third larval instar salivary glands. Degradation concurs with the transient increase of ribonucleolytic activities in the gland cells. In vitro nuclease assays using crude cytoplasmic extracts of ecdysone-treated salivary glands demonstrate degradation to be deadenylation-independent and that the induced ribonucleolytic activities initiate the degradation of the Lgp-1 transcripts in putative single-stranded loop regions. The independence of degradation from deadenylation is also found in vivo in transformed D. melanogaster carrying a modified Lgp-1 gene.

Unique structural and stabilizing roles for the individual pseudouridine residues in the 1920 region of Escherichia coli 23S rRNA.

The synthesis of a 5'-O-BzH-2'- O -ACE-protected pseudouridine phosphoramidite is reported [BzH, benzhydryloxy-bis(trimethylsilyloxy)silyl; ACE, bis(2-acetoxyethoxy)methyl]. The availability of the phosphoramidite allows for reliable and efficient syntheses of hairpin RNAs containing single or multiple pseudouridine modifications in the stem or loop regions. Five 19-nt hairpin RNAs representing the 1920-loop region (G(1906)-C(1924)) of Escherichia coli 23S rRNA were synthesized with pseudouridine residues located at positions 1911, 1915 and 1917. Thermodynamic parameters, circular dichroism spectra and NMR data are presented for all five RNAs. Overall, three different structural contexts for the pseudouridine residues were examined and compared with the unmodified RNA. Our main findings are that pseudouridine modifications exhibit a range of effects on RNA stability and structure, depending on their locations. More specifically, pseudouridines in the single-stranded loop regions of the model RNAs are slightly destabilizing, whereas a pseudo-uridine at the stem-loop junction is stabilizing. Furthermore, the observed effects on stability are approximately additive when multiple pseudouridine residues are present. The possible relationship of these results to RNA function is discussed.

Cleavage properties of an estrogen-regulated polysomal ribonuclease involved in the destabilization of albumin mRNA.

Previous work from this laboratory [Dompenciel,R.E., Garnepudi,V.R. and Schoenberg,D.R. (1995)J. Biol. Chem.270, 6108-6118] described the purification and properties of an estrogen-regulated endonuclease isolated from Xenopus liver polysomes that is involved in the destabilization of albumin mRNA. The present study mapped cleavages made by this enzyme onto the secondary structure of the portion of albumin mRNA bearing the major cleavage sites. The predominant cleavages occur in the overlapping APyrUGA sequence AUUGACUGA present in a single-stranded loop region, and in AUUGA located within a bulged AU-rich stem. A structural mutation which converted the major loop cleavage site to a hairpin bearing one APyrUGA element eliminated cleavage at the intact site. This confirms that the polysomal RNase is specific for single-stranded RNA. Additional point mutations in the major loop characterized the nucleoside sequence requirements for cleavage. Finally, snake venom exonuclease was used to demonstrate the polysomal RNase generates products with a 3' hydroxyl. Binding of an estrogen-induced protein to a portion of the 3'UTR of vitellogenin mRNA may be involved in its stabilization by estrogen [Dodson,R.E. and Shapiro,D.J. (1994)Mol. Cell. Biol.14, 3130-3138]. The core binding site for this protein bears the sequence APyrUGA, suggesting that stabilization may be accomplished by occlusion of a cleavage site for the polysomal RNase.

RNA structure at high resolution.

Studies of RNA structural motifs at high resolution by NMR and X-ray crystallographic methods have provided many insights into the fundamental forces that give rise to the unique structural characteristics of RNA. Non-Watson-Crick purine-pyrimidine, purine-purine, and pyrimidine-pyrimidine base pairing, as well as base-phosphate and base-ribose hydrogen bonding, are important forces for folding and stabilizing RNA structures. Base stacking is as important in determining RNA conformations as hydrogen bonding interactions. With the noncanonical interactions, many single-stranded loop regions such as hairpin loops, bulge loops, and internal loops fold into well-defined secondary structures. Loop-loop and loop-helix interactions can produce tertiary structures such as pseudoknots. Also, single strands adjacent to helical regions can form tertiary contacts with base-paired nucleotides of the helices. As we learn more about the structures of the important motifs we can ask more specific questions about the mechanisms of RNA-mediated functions. Conformational flexibility rather than a specific shape of the RNA may be important for some biological reactions. However, knowledge of the structures and the ease of conformational change of the molecules involved in any process are essential for understanding and eventually controlling the process.

Enediyne-mediated cleavage of RNA

RNA cleavage by enediyne anticancer antibiotics was shown to occur with no apparent sequence selectivity, but RNA structure appears to be important in those substrates where cleavage was observed. Neocarzinostatin (NCS) cleaved a wider variety of RNA substrates than either esperamicin (ESP) or calicheamicin (CAL), and dynemicin (DYN) has yet to cleave any RNA substrate tried. NCS, ESP, and CAL were all observed to cleave RNA substrates near the 5′-end, and all three compounds exhibited cleavage in single-stranded loop regions of the RNA substrates. NCS required no thiol for activation and subsequent cleavage, but ESP and CAL required addition of thiol, as expected, for cleavage to occur. An RNA hairpin substrate containing a UCCU sequence, equivalent to the TCCT sequence preferred by CAL in double-stranded DNA substrates, was cleaved by CAL, but no retention of selectivity for the UCCU site was retained by CAL in this RNA substrate. This study confirms an earlier observation that RNA is a substrate for enediyne cleavage, and indicates that nucleic acid cleaving compounds such as the enediynes could be useful probes of RNA three-dimensional structure.

Visualisation of inter loop tertiary base pairing and stacking interactions in an unusual slipped loop DNA structure

The conformation of an unusual slipped loop DNA structure exhibited by the sequence d(GAATTCCCGAATTC)2 is determined using a combination of geometrical and molecular mechanics methods. This sequence is known to form a B-DNA-like duplex with the central non-complementary cytosines extruded into single stranded loop regions. The unusual feature is that the interior guanine does not pair with the cytosine across, instead, it pairs with the cytosine upstream by skipping two cytosines, leading to a slipped loop DNA structure with the loops staggered by two base pairs. The two loops, despite being very small, can fold across minor or major groove symmetrically or asymmetrically disposed, with one of the loop bases partially blocking the major or minor groove. Most interestingly, for certain conformations, the loop bases approach one another at close proximity so as to engage even in base pairing as well as base stacking interactions across the major groove. While such pairing and stacking are common in the tertiary folds of RNA, this is the first time that such an interaction is visualized in a DNA. This observation demonstrates that a W-C pair can readily be accomplished in a typical slipped loop structure postulated for DNA. Such tertiary loop interaction may prevent access to regulatory proteins across the major groove of the duplex DNA, thus providing a structure-function relation for the occurrence of slipped loop structure in DNA.

Deletion of internal sequence on the HDV-ribozyme: elucidation of functionally important single-stranded loop regions.

In elucidating functionally important single-stranded loop regions derived mainly from three models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several internal deletion variants of the HDV133 molecule (654-786 nt on genomic RNA) by oligonucleotide-directed mutagenesis. When self-cleavage activities were compared among variants, the HDV133DI-1 (deletion of 701-718 nt) and HDV133DI-3 (deletion of 740-752 nt) ribozyme could maintain their self-cleavage activity, despite at reduced level. However, the activity could be regained in both mutants by some extent under partially denaturing conditions. These results suggest that the above two single-stranded RNA loop regions in HDV ribozyme are not part of the catalytic core but might be involved in the stability of the molecule. In contrast, deletion mutants such as HDV133DI-2 (deletion of 696-722 nt), HDV88DI-1 (deletion of 701-718 nt), HDV88DI-2 (deletion of 696-722 nt), and HDV88DI-4 (deletion of 733-760 nt) abolished catalytic activity. These results suggest that the remaining single-stranded regions of bases between 726-731 and 762-766 in the HDV88 ribozyme may be the potential regions to interact with Mg2+ ions.

Dam methyltransferase sites located within the loop region of the oligopurine-oligopyrimidine sequences capable of forming H-DNA are undermethylatedin vivo

Several derivatives of pUC18 plasmid were constructed that contained oligopurine-oligopyrimidine (pur-pyr) motifs surrounded by Dam methylation sites. Inserts of two of the molecules (pPP1 and pPP2) were able to adopt the triple-stranded conformation in vitro and show in vivo a remarkable undermethylation of specific sites when grown in JM105 dam+ strain. Mapping experiments revealed that undermethylated GATC sequences were located exclusively within the single-stranded loop region of the sequence involved in H-DNA formation. Control molecules which either contained the pur-pyr tracts (pPPK and pKK42) or not (pUC18) and were not able to form the triple-stranded conformation were found to be normally methylated by the dam gene product in vivo. Location of GATC within the triplex forming sequence seems to be a prerequisite for achieving its in vivo undermethylation. E.coli host factors are involved in the observed phenomenon. This has been deduced from the fact that the undermethylated state of pPP1 and pPP2 does not depend on the phase of growth of host cells and is steadily maintained up to 50 hours, whereas the kinetics of Dam methylation in vitro of sites located within the triplex loop does not differ substantially from the kinetics of methylation of other sites on the vector. Full methylation can be readily achieved in vitro. Additional factor(s) that operate in vivo to control the undermethylated state are most likely proteins since the observed effect can be suppressed by chloramphenicol administration to the cell cultures.