Triple Helix-forming Oligonucleotides Research Articles

CFTR and dystrophin encoding plasmids carrying both luciferase reporter gene, nuclear import specific sequences and triple helix sites

Duchenne Muscular Dystrophy and Cystic Fibrosis are two major monogenetic diseases which could be treated by non-viral gene therapy. For this purpose, plasmid DNA (pDNA) coding for the functional genes requires its equipment with signal molecules favouring its intracellular trafficking and delivery in the nucleus of the target cells. Here, two novel constructions of large pDNAs encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and full-length dystrophin (DYS) genes are reported. The expression of CFTR and DYS genes are driven respectively by the hCEF1 airway epithelial cells and spc5–12 muscle cells specific promoter. Those pDNAs encode also the luciferase reporter gene driven by the CMV promoter to evaluate gene delivery in animals by bioluminescence. In addition, oligopurine • oligopyrimidine sequences are inserted to enable equipment of pDNAs with peptides conjugated with a triple helix forming oligonucleotide (TFO). Furthermore, specific κB sequences are also inserted to promote their NFκB-mediated nuclear import. pDNA constructions are reported; transfection efficiency, tissue specific expression of CFTR and dystrophin in target cells, and triple helix formation are demonstrated. These plasmids are tools of interest to develop non-viral gene therapy of Cystic Fibrosis and Duchenne Muscular Dystrophy.

Versatile and efficient chromatin pull-down methodology based on DNA triple helix formation

The goal of present paper is to develop a reliable DNA-based method for isolation of protein complexes bound to DNA (Isolation of DNA Associated Proteins: IDAP). We describe a robust and versatile procedure to pull-down chromatinized DNA sequences-of-interest by formation of a triple helix between a sequence tag present in the DNA and a complementary triple helix forming oligonucleotide (TFO) coupled to a desthiobiotin residue. Following optimization to insure efficient recovery of native plasmids via TFO probe in vitro, the procedure is shown to work under various experimental situations. For instance, it allows capture proteins associated to plasmids hosted in E. coli, and is also successfully applied to recovering nucleosomes in vitro opening many possibilities to study post translational modifications of histones in a genuine nucleosome context. Incubation in human nuclear extracts of a plasmid carrying a NF-κB model promoter is shown to pull-down a specific transcription factor. Finally, isolation of a specific locus from human genomic chromatin has been successfully achieved (Chromatin-of-Interest Fragment Isolation: CoIFI). In conclusion, the methodology can be implemented for capturing proteins that specifically bind to any sequence-of-interest, DNA adduct or secondary structure provided a short sequence tag for triple helix formation is located nearby.

Recognition Mechanisms and Applications of Peptide Nucleic Acids Targeting Double-stranded DNA.

Targeting double-stranded DNA (dsDNA) with high affinity and specificity has become a hot topic in biochemistry and molecular biology research. Gene diagnosis and therapy, DNA manipulation, and gene expression regulation could be achieved based on certain recognition principles through specific interactions between dsDNA and natural nucleotides or synthesized ligands. Some ligands (e.g., peptide nucleic acids (PNAs), triple helix-forming oligonucleotides (TFOs), oligopolyamides, and zinc-finger peptides) show sufficient affinity and specificity for targeting dsDNA sequences. PNA can simply synthesize and recognize dsDNA without sequence limitation under physiological conditions. This paper provides a review on the recognition mechanisms, influencing factors, and applications of the four recognition modes of PNA targeting dsDNA. These modes include triplex invasion, triplex binding, duplex invasion, and double duplex invasion. This paper also discusses the challenges to be addressed by future research to fully explore the potential of PNA probe design for specific dsDNA recognition.

Aptamer based lysozyme assay using fluorescent CuInS2 quantum dots and graphene oxide, and its application to inhibitor screening

We report on a widely applicable approach for protein detection by using triple-helix DNA mediated CuInS2 quantum dot (QD) and graphene oxide (GO) nanocomposite. The CuInS2 QDs were coated with mercaptopropionic acid and then covalently linked to a hairpin aptamer against lysozyme (HLA). Single-stranded DNA (triple helix-forming oligonucleotide; THFO) readily absorbs on the surface of GO via π-stacking interaction, and this results in the formation of THFO-GO. If HLA-CuInS2 QDs are added to the THFO-GO system, the fluorescence of HLA-CuInS2 QDs (at excitation/emission wavelengths of 590/665 nm) is quenched. Lysozyme has a higher affinity for HLA than THFO. Therefore, in the presence of lysozyme, it will bind to the HLA-CuInS2 QD and displace the THFO-GO. This results in the restoration of fluorescence that is related to the concentration of lysozyme. The fluorescence of the QDs is turned on. The calibration plot is linear in the 0.01 to 1.8 ng·mL‾1 concentration range, with a 3 pg·mL‾1 detection limit (at a signal-to-noise ratio of 3). The method was also applied to study the inhibition of lysozyme by Ivy Ec . In our perception, this method has a wide scope in that it may become applicable to any protein for which an appropriate aptamer is available.

In VitroAntiviral Activity of Circular Triple Helix Forming Oligonucleotide RNA towards Feline Infectious Peritonitis Virus Replication

Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log10 from 1014 in the virus-inoculated cells to 109 in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection.

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.

Catalytic Activity Control of Restriction Endonuclease—Triplex Forming Oligonucleotide Conjugates

Targeting of individual genes in complex genomes requires endonucleases of extremely high specificity. To direct cleavage at the unique site(s) in the genome, both naturally occurring and artificial enzymes have been developed. These include homing endonucleases, zinc-finger nucleases, transcription activator-like effector nucleases, and restriction or chemical nucleases coupled to a triple-helix forming oligonucleotide (TFO). The desired cleavage has been demonstrated both in vivo and in vitro for several model systems. However, to limit cleavage strictly to unique sites and avoid undesired reactions, endonucleases with controlled activity are highly desirable. In this study we present a proof-of-concept demonstration of two strategies to generate restriction endonuclease-TFO conjugates with controllable activity. First, we combined the restriction endonuclease caging and TFO coupling procedures to produce a caged MunI-TFO conjugate, which can be activated by UV-light upon formation of a triple helix. Second, we coupled TFO to a subunit interface mutant of restriction endonuclease Bse634I which shows no activity due to impaired dimerization but is assembled into an active dimer when two Bse634I monomers are brought into close proximity by triple helix formation at the targeted site. Our results push the restriction endonuclease-TFO conjugate technology one step closer to potential in vivo applications.

Fluorescence Detection of Single‐Nucleotide Polymorphism with Single‐Strand Triplex‐Forming DNA Probes

Triple-helix-forming oligonucleotides (TFOs) are widespread in the genome and have been found in regulatory regions, especially in promoter zones and recombination hotspots of DNA. To specifically detect these polypurine sequences, we designed and synthesized two dual pyrene-labeled single-strand oligonucleotide probes (TFO-FPs) consisting of recognition, linker, and detection sequences. The hybridization processes of TFO-FPs with target polypurine oligonucleotides involve both Watson-Crick and Hoogsteen base-pairings. Through double sensing of oligonucleotide sequences, single mutations of target oligonucleotides are detected by monitoring changes in pyrene fluorescence. The high specificities of the probes are maintained over a wide temperature range without sacrifice of hybridization kinetics.

Targeting of an interrupted polypurine:polypyrimidine sequence in mammalian cells by a triplex-forming oligonucleotide containing a novel base analogue.

The DNA triple helix consists of a third strand of nucleic acid lying in the major groove of an intact DNA duplex. The most stable triplexes form on polypurine:polypyrimidine sequences, and pyrimidine interruptions in the purine strand are destabilizing. Sequence stringency is imparted by specific Hoogsteen hydrogen bonds between third strand bases and the purine bases in the duplex. Appropriate base and sugar modifications of triple helix-forming oligonucleotides (TFOs) confer chromosome targeting activity in living cells. However, broad utilization of TFOs as gene targeting reagents in mammalian cells has been limited by the requirement for homopurine target sequences. Although there have been a number of base analogues described that appear to be promising as candidates for triplex target expansion, none has been examined in a biological system. We have employed a postsynthetic strategy to prepare a collection of TFOs with base analogues at a defined position. Following assessment of affinity for a triplex target with a single C:G inversion, TFOs with a second generation of analogues were synthesized. One of these, TFO-5a, with 2'-OMe-guanidinylethyl-5-methylcytosine at the position corresponding to the C:G interruption in the target sequence, was further modified to confer bioactivity. The activity of this TFO, linked to psoralen, was measured in a mammalian cell line that was engineered by directed sequence conversion to carry a triplex target with a single C:G interruption. TFO-5a was active against this target and inactive against the corresponding target with an uninterrupted polypurine:polypyrimidine sequence.

Inhibition of human prostate cancer xenograft growth by 125I labeled triple-helin forming oligonucleotide directed against androgen receptor

The failure of hormone treatment for advanced prostate cancer might be related to aberrant activation of the androgen receptor. We have shown that (125)I labeled triple-helix forming oligonucleotide (TFO) against the androgen receptor gene inhibits androgen receptor expression and cell proliferation of LNCaP prostate cancer cells in vitro. This study aimed at exploring the effects of the (125)I-TFO on prostate tumor growth in vivo using a nude mouse xenograft model. TFO was labeled with (125)I by the iodogen method. Thirty-two nude mice bearing LNCaP xenograft tumors were randomized into 4 groups and were intratumorally injected with (125)I-TFO, unlabeled TFO, Na(125)I and normal saline. Tumor size was measured weekly. The tumor growth inhibition rate (RI) was calculated by measurement of tumor weight. The expression of the androgen receptor gene was performed by RT-PCR and immunohistochemical study. The prostate specific antigen (PSA) serum levels were measured by enzyme linked immunosorbent assay. The tumor cell apoptosis index (AI) was detected by TUNEL assay. Tumor measurements showed that tumor development was significantly inhibited by either (125)I-TFO or TFO, with tumor RIs of 50.79% and 32.80% respectively. (125)I-TFO caused greater inhibition of androgen receptor expression and higher AIs in tumor tissue than TFO. Both the tumor weight and the PSA serum levels in (125)I-TFO treated mice ((0.93 +/- 0.15) g and (17.43 +/- 1.85) ng/ml, respectively) were significantly lower than those ((1.27 +/- 0.21) g and (28.25 +/- 3.41) ng/ml, respectively) in TFO treated mice (all P < 0.05). Na(125)I did not significantly affect tumor growth and androgen receptor expression in tumor tissue. The (125)I-TFO can effectively inhibit androgen receptor expression and tumor growth of human prostate cancer xenografts in vivo. The inhibitory efficacy of (125)I-TFO is more potent than that of TFO, providing a reference for future studies of antigen radiotherapy.

Effects of anti-gene and antisense therapeutics on human prostate cancer xenograft in nude mice

To investigate the effects of triple-helix forming oligonucleotide (TFO) and antisense oligonucleotide (ASO) on androgen receptor (AR) expression and tumor growth of human prostate cancer xenografts in nude mice. Thirty-two nude mice were inoculated with human prostate cancer cells of the line LNCaP-C4-2 were randomized into 4 equal groups: TFO treatment group, undergoing intra-tumor injection of TFO at the dose of 25 mgxkg(-1)x(2d)(-1) for 14 times, ASO treatment group, undergoing intra-tumor injection of ASO at the same dose for 14 times, SCO group, undergoing intra-rumor injection of sequence control oligonucleotide (SCO) at the same dose for 14 times, and control group. The body weight and xenograft tumor volume of the nude mice were monitored during the therapy. 28 days later venous blood samples were collected to measure the level of prostate specific antigen (PSA) by radioimmunoassay and then the mice were killed with their tumors taken out to measure the weight, and RT-PCR, immunohistochemistry, and radioligand binding assay were used to detect the AR gene mRNA and protein expression in the tumor tissues. By the end of experiment the volumes and weights of tumor of the ASO and ASO groups were all significantly lower than those of the control group (all P < 0.01) with the inhibition rates of 67.55% and 41.06% respectively, and the volumes and weights of tumor of the TFO group were all significantly lower than those of the ASO group (all P < 0.05). The tumor weight and AR expression levels of TFO group were significantly lower than those of ASO group (P < 0.05). The serum PSA level of TFO group was (6.6 +/- 1.0) ng/ml, significantly lower than that of the ASO group [(19.8 +/- 3.7) ng/ml, P < 0.05]. The mRNA and protein expression levels of AR of the TFO group were significantly lower than those of the other groups (all P < 0.05). There were no significant differences in all the above mentioned markers between the SCO and control groups (all P > 0.05). TFO shows significantly higher inhibitory effect on AR expression and tumor growth of human prostate cancer xenograft than ASO, and is a promising agent for prostate cancer gene therapy.

Targeted Gene Knock In and Sequence Modulation Mediated by a Psoralen-linked Triplex-forming Oligonucleotide*

Information from exogenous donor DNA can be introduced into the genome via homology-directed repair (HDR) pathways. These pathways are stimulated by double strand breaks and by DNA damage such as interstrand cross-links. We have employed triple helix-forming oligonucleotides linked to psoralen (pso-TFO) to introduce a DNA interstrand cross-link at a specific site in the genome of living mammalian cells. Co-introduction of duplex DNA with target region homology resulted in precise knock in of the donor at frequencies 2-3 orders of magnitude greater than with donor alone. Knock-in was eliminated in cells deficient in ERCC1-XPF, which is involved in recombinational pathways as well as cross-link repair. Separately, single strand oligonucleotide donors (SSO) were co-introduced with the pso-TFO. These were 10-fold more active than the duplex knock-in donor. SSO efficacy was further elevated in cells deficient in ERCC1-XPF, in contrast to the duplex donor. Resected single strand ends have been implicated as critical intermediates in sequence modulation by SSO, as well as duplex donor knock in. We asked whether there would be a competition between the donor species for these ends if both were present with the pso-TFO. The frequency of duplex donor knock in was unaffected by a 100-fold molar excess of the SSO. The same result was obtained when the homing endonuclease I-SceI was used to initiate HDR at the target site. We conclude that the entry of double strand breaks into distinct HDR pathways is controlled by factors other than the nucleic acid partners in those pathways.

Single-molecule observations of topotecan-mediated TopIB activity at a unique DNA sequence

The rate of DNA supercoil removal by human topoisomerase IB (TopIB) is slowed down by the presence of the camptothecin class of antitumor drugs. By preventing religation, these drugs also prolong the lifetime of the covalent TopIB–DNA complex. Here, we use magnetic tweezers to measure the rate of supercoil removal by drug-bound TopIB at a single DNA sequence in real time. This is accomplished by covalently linking camptothecins to a triple helix-forming oligonucleotide that binds at one location on the DNA molecule monitored. Surprisingly, we find that the DNA dynamics with the TopIB–drug interaction restricted to a single DNA sequence are indistinguishable from the dynamics observed when the TopIB–drug interaction takes place at multiple sites. Specifically, the DNA sequence does not affect the instantaneous supercoil removal rate or the degree to which camptothecins increase the lifetime of the covalent complex. Our data suggest that sequence-dependent dynamics need not to be taken into account in efforts to develop novel camptothecins.

Targeted gene modification by oligonucleotides and small DNA fragments in eukaryotes

Targeted modification of genomic DNA (gDNA) in a cell permanently changes its phenotype. Numerous methods to modify target portions of gDNA in eukaryotes have been developed, and several have found practical application (e.g., homologous recombination-utilized modification in mouse ES cells). The past decade has seen active research both in oligonucleotide-directed modifications (where the oligonucleotides can be chimeric RNA/DNA oligonucleotides, single-stranded oligonucleotides, or triple helix-forming oligonucleotides), and in small DNA fragment-directed targeted modifications. These modifications can alter gDNA at several basepairs. In order to specifically direct the reaction to the targeted site, short oligonucleotides (shorter than 80-mers) or small DNA fragments (500 to approximately 2000 bps) are needed. Such oligonucleotides and small DNA fragments can be prepared easily. Although the mechanism by which targeted modification occurs has not been fully elucidated, numerous eukaryotic target genes have been modified using these methods in both cultured cells and live animals. The effectiveness of each method has been confirmed, but the efficiency of modification for all these approaches is presently insufficient for practical use. Further studies, including elucidation of complete mechanisms and optimization of methods for introducing oligonucleotides or small DNA fragment into cell nuclei, are required.

Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids

Sequence-specific DNA-binding molecules such as triple helix-forming oligonucleotides (TFOs) provide a means for inducing site-specific mutagenesis and recombination at chromosomal sites in mammalian cells. However, the utility of TFOs is limited by the requirement for homopurine stretches in the target duplex DNA. Here, we report the use of pseudo-complementary peptide nucleic acids (pcPNAs) for intracellular gene targeting at mixed sequence sites. Due to steric hindrance, pcPNAs are unable to form pcPNA–pcPNA duplexes but can bind to complementary DNA sequences by Watson–Crick pairing via double duplex-invasion complex formation. We show that psoralen-conjugated pcPNAs can deliver site-specific photoadducts and mediate targeted gene modification within both episomal and chromosomal DNA in mammalian cells without detectable off-target effects. Most of the induced psoralen-pcPNA mutations were single-base substitutions and deletions at the predicted pcPNA-binding sites. The pcPNA-directed mutagenesis was found to be dependent on PNA concentration and UVA dose and required matched pairs of pcPNAs. Neither of the individual pcPNAs alone had any effect nor did complementary PNA pairs of the same sequence. These results identify pcPNAs as new tools for site-specific gene modification in mammalian cells without purine sequence restriction, thereby providing a general strategy for designing gene targeting molecules.

Photoinduced Crosslinking of Double-helical DNA by Psoralen Covalently Linked to a Triple Helix-forming Oligonucleotide under Near-physiological Conditions

Stable triplexes have been generated under near-physiological conditions by the introduction of the C and T base analogues 3-methyl-2-aminopyridine-2 ′-deoxyriboside and 5-(3-aminoprop-2-ynyl)-′-deoxyuridine into psoralen-conjugated triplex-forming oligonucleotides. After irradiation with UV light at 365 nm, photo-induced cross-linking of the TFO to double-helical DNA was observed by UV-melting analysis and fluorescence measurements.

Extensive Sugar Modification Improves Triple Helix Forming Oligonucleotide Activity in Vitro but Reduces Activity in Vivo

We are developing triple helix forming oligonucleotides (TFOs) for gene targeting. Previously, we synthesized bioactive TFOs containing 2'-O-methylribose (2'-OMe) and 2'-O-aminoethylribose (2'-AE) residues. Active TFOs contained four contiguous 2'-AE residues and formed triplexes with high thermal stability and rapid association kinetics. In an effort to further improve bioactivity, we synthesized three series of TFOs containing the 2'-AE patch and additional ribose modifications distributed throughout the remainder of the oligonucleotide. These were either additional 2'-AE residues, the conformationally locked BNA/LNA ribose with a 2'-O,4'-C-methylene bridge, or the 2'-O,4'-C-ethylene analogue (ENA). The additionally modified TFOs formed triplexes with greater thermal stability than the reference TFO, and some had improved association kinetics. However, the most active TFOs in the biochemical and biophysical assays were the least active in the bioassay. We measured the thermal stability of triplexes formed by the TFOs in each series on duplex targets containing a change in sequence at a single position. The Tm value of the variant sequence triplexes increased as the number of all additional modifications increased. A simple explanation for the failure of the improved TFOs in the bioassay was that the increased affinity for nonspecific targets lowered the effective nuclear concentration. Enhancement of TFO bioactivity will require chemical modifications that improve interaction with the specific targets while retaining selectivity against mismatched sequences.

A human neuronal tissue culture model for Lesch‐Nyhan disease

Mutations in the gene encoding the purine salvage enzyme, hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause Lesch-Nyhan disease, a neurodevelopmental disorder characterized by cognitive, neurological, and behavioral abnormalities. Despite detailed knowledge of the enzyme's function, the key pathophysiological changes that accompany loss of purine recycling are unclear. To facilitate delineating the consequences of HPRT deficiency, four independent HPRT-deficient sublines of the human dopaminergic neuroblastoma, SK-N-BE(2) M17, were isolated by targeted mutagenesis with triple helix-forming oligonucleotides. As a group, these HPRT-deficient cells showed several significant abnormalities: (i) impaired purine recycling with accumulation of hypoxanthine, guanine, and xanthine, (ii) reduced guanylate energy charge and GTP:GDP ratio, but normal adenylate energy charge and no changes in any adenine nucleotide ratios, (iii) increased levels of UTP and NADP+, (iv) reduced DOPA decarboxylase, but normal monoamines, and (v) reduction in cell soma size. These cells combine the analytical power of multiple lines and a human, neuronal origin to provide an important tool to investigate the pathophysiology of HPRT deficiency.

Regulation of Transcription by Synthetic DNA‐Bending Agents

Gene expression is regulated by a complex interplay between binding and the three-dimensional arrangement of transcription factors with RNA polymerase and DNA. Previous studies have supported a direct role for DNA bending and conformation in gene expression, which suggests that agents that induce bends in DNA might be able to control gene expression. To test this hypothesis, we examined the effect of triple-helix-forming oligonucleotide (TFO) bending agents on the transcription of luciferase in an in vitro transcriptional/translational system. We find that transcription is regulated only by a TFO that induces a bend in the DNA. Related TFOs that do not induce bends in DNA have no effect on transcription. Reporter expression can be increased by as much as 80 % or decreased by as much as 50 % depending on the phasing of the upstream bend relative to the promoter. We interpret the results as follows: when the bend is positioned such that the upstream DNA is curved toward the RNA polymerase on the same DNA face, transcription is enhanced. When the upstream DNA is curved away, transcription is attenuated. These results support the hypothesis that DNA-bending agents might have the capability to regulate gene expression, thereby opening up a previously undervalued avenue in research on the artificial control of gene expression.

Targeted Cross-linking of the Human β-Globin Gene in Living Cells Mediated by a Triple Helix Forming Oligonucleotide

Triple helix forming oligonucleotides (TFOs) may have utility as gene targeting reagents for "in situ" gene therapy of genetic disorders. Triplex formation is challenged by negative charge repulsion between third strand and duplex phosphates, and destabilizing positive charge repulsion between adjacent protonated cytosines within pyrimidine motif third strands. Here we describe the synthesis of TFOs designed to target a site in the human beta-globin gene, which is the locus for mutations that underlie the beta-globinopathies, including sickle cell anemia. The target is an uninterrupted polypurine:polypyrimidine sequence, containing four adjacent cytosines, next to a psoralen cross-link site. Pyrimidine motif TFOs that contained four adjacent cytosines or 5-methylcytosines did not form stable triplexes at physiological pH, despite the introduction of otherwise stabilizing base and sugar analogues. We synthesized a series of pso-TFOs containing 2'-O-methyl (OMe) and 2'-O-aminoethoxy substitutions (AE), as well as 8-oxo-adenine (A8) and 2'-O-methylpseudoisocytidine (P) as neutral cytosine replacements. Thermal stability measurements indicated that TFOs with A8 did not meet criteria established in previous work. However, TFOs with P did form triplexes with appropriate T(m) and k(ON) values. A pso-TFO with AE and P residues was sufficiently active to permit the determination of targeting in living cells by direct measurement of cross-link formation at the target site. Our results validate the modification format described in our previous studies and indicate that P substitutions are an effective solution to the problem of targeting genomic sequences containing adjacent cytosines.