DNA Triplex Formation Research Articles

Selective inhibition of the human tie-1 promoter with triplex-forming oligonucleotides targeted to Ets binding sites.

The Tie receptors (Tie-1 and Tie-2/Tek) are essential for angiogenesis and vascular remodeling/integrity. Tie receptors are up-regulated in tumor-associated endothelium, and their inhibition disrupts angiogenesis and can prevent tumor growth as a consequence. To investigate the potential of anti-gene approaches to inhibit tie gene expression for anti-angiogenic therapy, we have examined triple-helical (triplex) DNA formation at 2 tandem Ets transcription factor binding motifs (designated E-1 and E-2) in the human tie-1 promoter. Various tie-1 promoter deletion/mutation luciferase reporter constructs were generated and transfected into endothelial cells to examine the relative activities of E-1 and E-2. The binding of antiparallel and parallel (control) purine motif oligonucleotides (21-22 bp) targeted to E-1 and E-2 was assessed by plasmid DNA fragment binding and electrophoretic mobility shift assays. Triplex-forming oligonucleotides were incubated with tie-1 reporter constructs and transfected into endothelial cells to determine their activity. The Ets binding motifs in the E-1 sequence were essential for human tie-1 promoter activity in endothelial cells, whereas the deletion of E-2 had no effect. Antiparallel purine motif oligonucleotides targeted at E-1 or E-2 selectively formed strong triplex DNA (K(d) approximately 10(-7) M) at 37 degrees C. Transfection of tie-1 reporter constructs with triplex DNA at E-1, but not E-2, specifically inhibited tie-1 promoter activity by up to 75% compared with control oligonucleotides in endothelial cells. As similar multiple Ets binding sites are important for the regulation of several endothelial-restricted genes, this approach may have broad therapeutic potential for cancer and other pathologies involving endothelial proliferation/dysfunction.

Stabilization of purine motif DNA triplex by a tetrapeptide from the binding domain of HMGBI protein

DNA triple helix has attracted a lot of attention in the recent past because of their potential therapeutic applications to inhibit the transcription of genes associated with several human diseases. Therefore, studies on DNA triplex formation and stabilization have become very essential to understand the nature of DNA triplex both in quantitative and qualitative terms. We have studied the effect of a tetrapeptide, Pro-Lys-Arg-Trp (PKRW), containing the conserved sequence (PKR) of high mobility group (HMG) B1 DNA binding domain on the stability of purine motif DNA triplex. The triple stranded DNA complex (23R*R•Y) containing (GAA) repeats used in the present study is formed by: 5′-TCGCGAAGAAGAAGAAGAACGCT-3′, 5′-AGCGTTCTTCTTCTTCTTCGCGA-3′ and the third strand 5′-AAGAAGAAGAAGAAG-3′. The DNA triplex 23R*R•Y and its complex with PKRW were investigated by DNA-melting, UV-absorption, circular dichroism and fluorescence spectroscopy. The increased stability of 23R*R•Y triplex in presence of PKRW is supported by the following observations; (i) a marked increase in triplex melting temperature (ii) significant increase in the ellipticity of the characteristic negative band at 210 nm and (iii) a large change in free energy, Δ Δ G (Δ G of triplex-peptide complex – Δ G of free triplex), –9.6 kcal mol –1 on complexation with PKRW. Based on the above observations and fluorescence quenching data, we suggest a two stage model for the binding of PKRW to 23R*R•Y in which (a) PKRW binds to the minor groove of the 23R*R•Y with lysine and arginine making electrostatic interactions with phosphates of DNA, which is followed by (b) intercalation of tryptophan side chain between the A*A•T base triplets. We conclude that PKRW preferentially stabilizes Hoogsteen base pair to the Watson–Crick base pair in purine motif 23R*R•Y DNA triplex. It is tempting to speculate that HMGB1 plays an important role in recognizing the DNA triplex structures like H-DNA in the gene, which may have biological implications with respect to chromatin structure and its function in the cell.

Studies on the Extension of Sequence-independence and the Enhancement of DNA Triplex Formation

A more elaborate sequence-independent triple-helix formation viability study was carried out and extended from a recombination-like triple-helical DNA motif of a previous study (J. Mol. Recognition 14, 122–139 (2001)). The intended triple-helix was formed by mixing one part of a DNA hairpin duplex and one part of a single (or third) strand identical to one of the duplex strands and complementary to the other strand. In contrast to the common purine and pyrimidine motifs in triple-stranded DNA, the strands of the recombination-like motif are not monotonously built from pyrimidine only, or purine only, in the sequence. The stability of the recombination-like motif triplexes with varying sequences was monitored by UV thermal melting curves. The results showed that the order of the stability of the R-form DNA base triads (J. Mol. Biol., 239, 181–200 (1994)) is G*(G ○ C) > C*(C ○ G) > A*(A ○ T) >T*(T ○ A) (the Watson-Crick base pair is denoted in the parentheses) in 200 mM NaCl, at pH 7. In an attempt to increase the stability of the triplex in the recombination-like motif, we replaced cytidine by 5-methylcytidine (mC) of the third strand. There is a general trend that mC modification stabilizes the complex (<2 °C per mC). The complex is furthermore stabilized by Mg2+ ion. The Tm increases from 7 to 2 °C from less stable to highly stable triplex by 20 mM Mg2+ ion in solution.

LacI‐mediated sequence‐specific affinity purification of plasmid DNA for therapeutic applications

Affinity purification of plasmid DNA is an attractive option for the biomanufacture of therapeutic plasmids, which are strictly controlled for levels of host protein, DNA, RNA, and endotoxin. Plasmid vectors are considered to be a safer alternative than viruses for gene therapy, but milligram quantities of DNA are required per dose. Previous affinity approaches have involved triplex DNA formation and a sequence-specific zinc finger protein. We present a more generically applicable protein-based approach, which exploits the lac operator, present in a wide diversity of plasmids, as a target sequence. We used a GFP/His-tagged LacI protein, which is precomplexed with the plasmid, and the resulting complex was immobilized on a solid support (TALON resin). Ensuing elution gives plasmid DNA, in good yield (>80% based on recovered starting material, 35-50% overall process), free from detectable RNA and protein and with minimal genomic DNA contamination. Such an affinity-based process should enhance plasmid purity and ultimately, after appropriate development, may simplify the biomanufacturing process of therapeutic plasmids.

Amino-functionalized DNA: the properties of C5-amino-alkyl substituted 2'-deoxyuridines and their application in DNA triplex formation

The incorporation of C5-amino-modified 2′-deoxyuridine analogues into DNA have found application in nucleic acid labelling, the stabilization of nucleic acid structures, functionalization of nucleic acid aptamers and catalysts, and the investigation of sequence-specific DNA bending. In this study, we describe the physicochemical properties of four different C5-amino-modified 2′-deoxyuridines in which the amino group is tethered to the base via a 3-carbon alkyl, Z- or E-alkenyl or alkynyl linker. Conformational parameters of the nucleosides and their pKa values were deduced using 1H NMR. All of them display the expected anti-conformation of the nucleoside with 2′-endo sugar puckers for the deoxyribose ring. A preference for the cisoid conformation for the Z-alkenyl analogue is found, while the E-alkenyl analogue exists exclusively as its transoid conformation. The pKa values range from 10.0 for the analogue with an aliphatic propyl linker to 8.5 for the propargylamino analogue. The analogues have been used for the synthesis of triple-helix forming oligonucleotides (TFOs) in which they replace thymidine in the natural sequence. Oligonucleotides containing the propargylamino analogue display the highest stability especially at low pH, while those containing analogues with propyl and especially Z-alkenyl linkers are destabilized to a great extent. TFOs containing the analogue with the E-alkenyl linker have stability similar to the unmodified structures. The chemical synthesis of TFOs containing the analogue, 5-(3-hydroxyprop-1-ynyl)-2′-deoxyuridine that possesses a neutral but polar side chain show a remarkable stability, which is higher than that of all TFOs containing the alkylamino or alkenylamino analogues and only slightly lower than that of TFOs containing the propargylamino analogue. Both the hydroxyl and propargylamino substitutions impart enhanced triple-helix stability relative to the analogous sequences containing C5-propynyl-2′-deoxyuridine. Furthermore, a similar dependence of stability on pH is found between TFOs containing the hydroxypropynyl modifications and those containing the propargylamino side chains. This suggests that the major factor responsible for stabilizing such triple helices is due to the presence of the alkyne with an attached electronegative group.

Antitumor Polycyclic Acridines. Part 16. Triplex DNA as a Target for DNA-Binding Polycyclic Acridine Derivatives

Triple-stranded DNA structures have been implicated in a number of major biological processes, including the transcription and translation of a number of genes, as well as in the interaction of DNA with a number of proteins. Furthermore, antigene therapies under development are based on the recognition and binding of a single oligonucleotide strand to a double-stranded sequence, thus forming a triple helix. Triplex DNA formation is a relatively weak and temporary phenomenon; therefore, molecules that selectively bind to and stabilize triple helices may show a variety of novel biological effects. The biophysical and biological characterization of a series of antitumor polycyclic acridines that bind to triplex DNA is reported. These compounds, whose synthesis has been previously reported, have been tested for their interaction with both purine and pyrimidine type triple helices and compared with the relevant double-stranded DNA. As a pyrimidine triplex model we have used the T*AT sequence, which we have compared with the AT duplex, whereas the purine triplex oligonucleotide d[G3A4G3]*d[G3A4G3].d[C3T4C3] has been compared with the duplex d[G3A4G3].d[C3T4C3]. The compounds demonstrate various degrees of preferential binding to triplex DNA over normal duplex DNA, as measured by UV, fluorescence, circular dichroism, and thermal denaturation. Tri-substituted acridine derivatives demonstrated the highest affinity and ability to stabilize triplex DNA structures. Furthermore, structure/affinity analysis gives insights into the structural features that optimize affinity and selectivity for triplex DNA, and may play a role in their profile of antitumor activity.

DNA binding and antigene activity of a daunomycin-conjugated triplex-forming oligonucleotide targeting the P2 promoter of the human c-myc gene.

Triplex-forming oligonucleotides (TFO) that bind DNA in a sequence-specific manner might be used as selective repressors of gene expression and gene-targeted therapeutics. However, many factors, including instability of triple helical complexes in cells, limit the efficacy of this approach. In the present study, we tested whether covalent linkage of a TFO to daunomycin, which is a potent DNA-intercalating agent and anticancer drug, could increase stability of the triple helix and activity of the oligonucleotide in cells. The 11mer daunomycin-conjugated GT (dauno-GT11) TFO targeted a sequence upstream of the P2 promoter, a site known to be critical for transcription of the c-myc gene. Band-shift assays showed that the dauno-GT11 formed triplex DNA with enhanced stability compared to the unmodified TFO. Band shift and footprinting experiments demonstrated that binding of dauno-GT11 was highly sequence-specific with exclusive binding to the 11 bp target site in the c-myc promoter. The daunomycin-conjugated TFO inhibited transcription in vitro and reduced c-myc promoter activity in prostate and breast cancer cells. The daunomycin-conjugated TFO was taken up by cells with a distinctive intracellular distribution compared to free daunomycin. However, cationic lipid-mediated delivery was required for enhanced cellular uptake, nuclear localization and biological activity of the TFO in cells. Dauno-GT11 reduced transcription of the endogenous c-myc gene in cells, but did not affect expression of non-target genes, such as ets-1 and ets-2, which contained very similar target sequences in their promoters. Daunomycin-conjugated control oligonucleotides unable to form triplex DNA with the target sequence did not have any effect in these assays, indicating that daunomycin was not directly responsible for the activity of daunomycin-conjugated TFO. Thus, attachment of daunomycin resulted in increased triplex stability and biological activity of the 11mer GT-rich TFO without compromising its specificity. These results encourage further testing of this approach to develop novel antigene therapeutics.

PKD1 intron 21: triplex DNA formation and effect on replication.

Although autosomal dominant polycystic kidney disease is transmitted in an autosomal dominant fashion, there is evidence that the pathophysiology of cystogenesis involves a second hit somatic mutation superimposed upon the inherited germline mutation within the renal tubule cells. The polypurine.polypyrimidine (Pu.Py) tract of PKD1 intron 21 may play a role in promoting somatic mutations. To better characterize this tract and to evaluate its potential to participate in mutagenesis, we investigated the thermodynamics of intramolecular triplex formation by 15 Pu.Py mirror repeat tracts from PKD1 intron 21 by 2D gel electrophoresis. We demonstrate that intramolecular triplexes form with modest superhelical tensions for all the tracts examined. Primer extension studies demonstrated significant polymerase arrest within the Pu.Py tracts in one direction of replication only. We found correlation between polymerization arrest and both the potential length of the triplex and superhelical tension of intramolecular triplex formation. The presence of a Pu.Py tract also led to a replication blockade and double-strand breakage using an SV40 in vitro replication assay with HeLa cell extracts. During DNA replication, the G-rich template of the PKD1 Pu.Py tracts may form a triplex structure with the nascent strand, thereby blocking replication and potentially leading to recombination and mutation.

Targeted gene delivery to sinusoidal endothelial cells: DNA nanoassociate bearing hyaluronan-glycocalyx.

Liver sinusoidal endothelial cells (SECs) possess unique receptors that recognize and internalize hyaluronic acid (HA). To develop a system for targeting foreign DNA to SECs, comb-type polycations having HA side chains were prepared by coupling HA to poly(L-lysine) (PLL). The HA-grafted-PLL copolymer (PLL-g-HA) thus formed was mixed with DNA in 154 mM NaCl to form soluble nanoassociates bearing hydrated hyaluronate shells. Agarose gel retardation assays revealed selective interaction of the PLL backbone with DNA despite the presence of polyanionic HA side chains. To determine whether the PLL-g-HA/DNA complexes were recognized by SEC HA receptors in vivo, we injected Wistar rats i.v. via the tail vein with PLL-g-HA complexed to a beta-galactosidase expression plasmid (pSV beta-Gal) labeled with 32P. One hour postinjection, >90% of the injected radioactivity remained in the liver. Administration of the PLL-g-HA complexed to an FITC-labeled DNA revealed that the carrier-DNA complex was distributed exclusively in SECs. A large number of SECs expressing beta-galactosidase was detected along the sinusoidal lining after transfection with PLL-g-HA/pSV beta-Gal. Moreover, PLL-g-HA effectively stabilized DNA triplex formation. In conclusion, the new PLL-g-HA/DNA carrier system permits targeted transfer of exogenous genes selectively to the SECs.

Synthesis and evaluation of a conformationally constrained pyridazinone PNA-monomer for recognition of thymine in triple-helix structures

A novel conformationally constrained pyridazinone E ag-base PNA-monomer 2 capable of binding thymine in a triplex motif was designed and synthesised. A bis-PNA with the E ag-base incorporated in the Hoogsteen strand was hybridised with a complementary DNA. Thermal stability studies revealed an increase in T m (4.3 °C per mod.) compared to a no-base unit, but showed no improvement over a previously described unconstrained analogue (E, 1). Surprisingly, no significant difference was found in the thermodynamic parameters (ΔH°, ΔS° and ΔG°) for PNA–DNA triplex formation involving 2 or the unconstrained analogue 1.

Use of laminar flow patterning for miniaturised biochemical assays

Laminar flow in microfluidic chambers was used to construct low (one dimensional) density arrays suitable for miniaturized biochemical assays. By varying the ratio of flows of two guiding streams flanking a sample stream, precise focusing and positioning of the latter was achieved, and reactive species carried in the sample stream were deposited on functionalized chip surfaces as discrete 50 microm wide lanes. Using different model systems we have confirmed the method's suitability for qualitative screening and quantification tasks in receptor-ligand assays, recording biotin-streptavidin interactions, DNA-hybridization and DNA-triplex formation. The system is simple, fast, reproducible, flexible, and has small sample requirements.

Positive Correlation Between DNA Polymerase α-Primase Pausing and Mutagenesis within Polypyrimidine/Polypurine Microsatellite Sequences

Microsatellite DNA sequences are ubiquitous in the human genome, and mutation rates of these repetitive sequences vary with respect to DNA sequence as well as length. We have analyzed polymerase-DNA interactions as a function of microsatellite sequence, using polypyrimidine/polypurine di- and tetranucleotide alleles representative of those found in the human genome. Using an in vitro primer extension assay and the mammalian DNA polymerase α-primase complex, we have observed a polymerase termination profile for each microsatellite that is unique to that allele. Interestingly, a periodic termination profile with an interval size (9–11 nucleotides) unrelated to microsatellite unit length was observed for the [TC] 20 and [TTCC] 9 templates. In contrast, a unit-punctuated polymerase termination profile was found for the longer polypurine templates. We detected strong polymerase pauses within the [TC] 20 allele at low reaction pH which were eliminated by the addition of deaza-dGTP, consistent with these specific pauses being a consequence of triplex DNA formation during DNA synthesis. Quantitatively, a strand bias was observed in the primer extension assay, in that polymerase synthesis termination is more intense when the polypurine sequence serves as the template, relative to its complementary polypyrimidine sequence. The HSV-tk forward mutation assay was utilized to determine the corresponding polymerase α-primase error frequencies and specificities at the microsatellite alleles. A higher microsatellite polymerase error frequency (50×10 −4 to 60×10 −4) was measured when polypurine sequences serve as templates for DNA synthesis, relative to the polypyrimidine template (18×10 −4). Thus, a positive correlation exists between polymerase α-primase pausing and mutagenesis within microsatellite DNA alleles.

Design and evaluation of novel nucleoside analogs (WNA) for specific formation of non-natural type triplexes containing a TA or CG interrupting site.

In a search of suitable recognition molecules for the formation of stable non-natural triplex DNA, we have developed the new nucleoside analogs (WNA, W-shape nucleoside analog) bearing an aromatic ring, a recognition base and a bicyclic skeleton to hold them. A successful result with WNA-betaT that recognizes a TA base pair selectively has suggested that the basic structure of the WNA is useful as a scaffold for search of other candidates for the formation of triplex containing a TA or a CG interrupting site. In this study, we have synthesized and investigated binding property of a variety of WNA candidates. As a result, we have determined new potential analog, WNA-betaC for a CG interrupting site. It should be noted that the triplexes containing a WNA-betaT:TA or a WNA-betaC:CG base triplet have exhibited higher stability than the natural anti-parallel triplexes constructed of A:AT and G:GC triplets. The results that H-WNA-betaT lacking a benzene ring did not show stabilization effect to any base pairs have clearly indicated that a benzene ring plays a key role in stability of triplexes.

Probing triplex formation by EPR spectroscopy using a newly synthesized spin label for oligonucleotides.

Spin labels have been extensively used to study the dynamics of oligonucleotides. Spin labels that are more rigidly attached to a base in an oligonucleotide experience much larger changes in their range of motion than those that are loosely tethered. Thus, their electron paramagnetic resonance spectra show larger changes in response to differences in the mobility of the oligonucleotides to which they are attached. An example of this is 5-(2,2,5,5-tetramethyl-3-ethynylpyrrolidine-1-oxyl)-uridine (1). How ever, the synthesis of this modified DNA base is quite involved and, here, we report the synthesis of a new spin-labeled DNA base, 5-(2,2,6,6-tetramethyl-4-ethynylpiperidyl-3-ene-1-oxyl)-uridine (2). This spin label is readily prepared in half the number of steps required for 1, and yet behaves in a spectroscopically analogous manner to 1 in oligonucleotides. Finally, it is shown here that both spin labels 1 and 2 can be used to detect the formation of both double-stranded and triplex DNA.

A molecular beacon strategy for real-time monitoring of triplex DNA formation kinetics.

We used a molecular beacon (MB) containing a 15-mer triplex-forming oligonucleotide (TFO) to probe in real-time the kinetics of triplex DNA formation in the left side of the TCl tract (502-516) of the c-src proto-oncogene in vitro. The metal ions Na+, K+, and Mg2+ stabilized triplex DNA at this site. The pseudo-first-order rate constant (kpsi) and the second-order association rate constant (k1) for the binding of the MB to the target duplex in 10 mM sodium phosphate buffer, pH 7.3, increased from 3.2 +/- 0.9 to 15 +/- 2.8 x 10(-3) s(-1) and 6.4 +/- 1.8 to 30 +/- 5.6 x 102 M(-1) s(-1), respectively, on increasing the MgCl2 concentration from 1 to 2.5 mM. Similar values were obtained for the triplex DNA stabilized by NaCl (100-250 mM). Surprisingly, the values were around 2 times higher in the presence of KCl. The AG of triplex formation in the presence of 1 mM MgCl2, 150 mM NaCl, and 150 mM KCl were -7.8 +/- 0.3, -8.2 +/- 0.3 and -8.7 +/- 0.7 kcal/mol respectively, despite significant differences in the values of deltaH and deltaS, suggesting enthalpy-entropy compensation in the stabilization of the triplex DNA by these metal ions. These results show the utility of MBs ih probing triplex DNA formation and in evaluating kinetic and thermodynamic parameters important for the design and development of TFOs as triplex DNA-based therapeutic agents.

Photoregulation of DNA triplex formation by azobenzene.

Formation and dissociation of DNA triplex are reversibly photoregulated by cis <--> trans isomerization of the azobenzene tethered to the third strand. When the azobenzene takes the trans from, a stable triplex is formed. Upon the isomerization of trans-azobenzene to its cis form by UV light irradiation (300 < lambda < 400 nm), however, the modified oligonucleotide is removed from the target duplex. The triplex is re-formed on photoinduced cis --> trans isomerization (lambda > 400 nm). The photoregulating activity significantly depends on the position of azobenzene in the third strand, as well as on the geometric position (meta or para) of its amido substituent. For m-amidoazobenzene, the photoregulation is the most effective when it is tethered to the 5'-end of the third strand. However, p-amidoazobenzene should be introduced into the middle of the strand for effective regulation. In the optimal cases, the change of T(m) of the triplex, caused by the cis <--> trans isomerization of azobenzene, is greater than 30 degrees C. UV-visible and CD spectroscopy, as well as computer modeling studies, clearly demonstrate that the trans-azobenzene intercalates between the base pairs in the target duplex and thus stabilizes the triplex by stacking interactions. On the other hand, nonplanar cis-azobenzene destabilizes the triplex due to its steric hindrance against the adjacent base pairs.

Equilibrium analysis of the efficiency of an autonomous molecular computer.

In the whiplash polymerase chain reaction (WPCR), autonomous molecular computation is implemented in vitro by the recursive, self-directed polymerase extension of a mixture of DNA hairpins. Although computational efficiency is known to be reduced by a tendency for DNAs to self-inhibit by backhybridization, both the magnitude of this effect and its dependence on the reaction conditions have remained open questions. In this paper, the impact of backhybridization on WPCR efficiency is addressed by modeling the recursive extension of each strand as a Markov chain. The extension efficiency per effective polymerase-DNA encounter is then estimated within the framework of a statistical thermodynamic model. Model predictions are shown to provide close agreement with the premature halting of computation reported in a recent in vitro WPCR implementation, a particularly significant result, given that backhybridization had been discounted as the dominant error process. The scaling behavior further indicates completion times to be sufficiently long to render WPCR-based massive parallelism infeasible. A modified architecture, PNA-mediated WPCR (PWPCR) is then proposed in which the occupancy of backhybridized hairpins is reduced by targeted PNA(2)/DNA triplex formation. The efficiency of PWPCR is discussed using a modified form of the model developed for WPCR. Predictions indicate the PWPCR efficiency is sufficient to allow the implementation of autonomous molecular computation on a massive scale.

A-tract DNA disfavours triplex formation

Optimisation of DNA triplex stability is of fundamental importance in the anti-gene strategy. In the present work, thermal denaturation studies by UV-spectrophotometry and structural and dynamical characterizations by NMR spectroscopy have been used systematically to investigate the effects on triplex stability of isolated insertions of different base triplets into an otherwise homogeneous 15-mer dT·dA-dT oligo-triplex. It is found that insertion of a single central C+·G-C or T·D-T triplet (D=2,6-diaminopurine) leads to a pronounced stabilization (up to 20 deg. C if the cytosine base is C5 methylated) at acidic as well as neutral pH. To a smaller degree, this is the case also for a C+·I-C triplet insertion.Using imino proton exchange measurements, it is shown that insertion of a DT base-pair in the underlying duplex perturbs the intrinsic A-tract structure in the same way as has been shown for a GC insert. We propose that the intrinsic properties of A-tract duplex DNA (e. g. high propeller twist and rigidity) are unfavourable for triplex formation and that GC- or DT-inserts stabilize the triplex by interfering with the A-tract features of the underlying duplex. The C+·I-C triplet without the N2 amino group in the minor groove is readily accommodated within the typical, highly propeller-twisted A-tract structure. This might be related to its smaller effect on the stability of the corresponding triplex.These results may be valuable for understanding DNA triplex formation in vivo as well as for the design of efficient triplex-forming oligonucleotides and in choosing suitable target sequences in the anti-gene strategy.

Molecular Beacons: Nucleic Acid Hybridization and Emerging Applications

Molecular beacons (MBs) are a novel class of nucleic acid probes that become fluorescent when bound to a complementary sequence. Because of this characteristic, coupled with the sequence specificity of nucleic acid hybridization and the sensitivity of fluorescence techniques, MBs are very useful probes for a variety of applications requiring the detection of DNA or RNA. We survey various applications of MBs, including the monitoring of DNA triplex formation, and describe recent developments in MB design that enhance their sensitivity.

Thermodynamic aspects of triplex DNA formation in crowded environments.

Thermodynamic aspects of triplex DNA formation in crowded environments.