Unmodified Oligonucleotides Research Articles

5‘-Modified G-Quadruplex Forming Oligonucleotides Endowed with Anti-HIV Activity: Synthesis and Biophysical Properties

Oligodeoxyribonucleotides of sequence d(5'TGGGAG3') carrying bulky aromatic groups at the 5' end were found to exhibit potent anti-HIV activity [Hotoda, H., et al. (1998) J. Med. Chem. 41, 3655-3663 and references therein]. Structure-activity relationship investigations indicated that G-quadruplex formation, as well as the presence of large aromatic substituents at the 5'-end, were both essential for their antiviral activity. In this work, we synthesized some representative examples of the anti-HIV active Hotoda's 6-mers and analyzed the resulting G-quadruplexes by CD, DSC, and molecular modeling studies, in comparison with the unmodified oligonucleotide. In the case of the sequence carrying the 3,4-dibenzyloxybenzyl (DBB) group, identified as the best candidate for further drug optimization, we developed an alternative protocol to synthesize the 5'-DBB-thymidine phosphoramidite building block in higher yields. The thermodynamic and kinetic parameters for the association/dissociation processes of the 5'-conjugated quadruplexes, determined with respect to the unmodified one, were discussed in light of the molecular modeling studies. The aromatic groups at the 5' position of d(5'TGGGAG3') dramatically enhance both the equilibrium and the rate of formation of the quadruplex complexes. The overall stability of the investigated quadruplexes was found to correlate with the reported IC50 values, thus furnishing quantitative evidence for the hypothesis that the G-quadruplex structures are the ultimate active species, effectively responsible for the biological activity.

Construction of microarrays for genotyping of DQA using unmodified 45-mer oligonucleotide

The human leukocyte antigen (HLA) class II system is strongly connected to immunological response and its compatibility between tissues is critical in transplantation. The simple robust typing analyses of HLA genes are extremely important. In this paper, we developed an approach based on microarray technology for genotyping of DQA gene. The microarrays were constructed with a total 31 unmodified 45-mer oligonucleotide. The second exon of DQA gene was amplified, and allowed to hybridize with the array. DQA genotypes were assigned by quantitative analysis of the hybridization results. The arrays were evaluated by DQA genotyping of nine reference samples and 120 clinical samples. The results demonstrate that the genotyping accuracy/concordance achieved 97.5% compared with the direct DNA sequencing. Although our methods did not perform high-resolution genotyping, it could be an alternative for serological typing in routine medical practice.

New Aspects of the Fragmentation Mechanisms of Unmodified and Methylphosphonate-Modified Oligonucleotides

A set of pentanucleotides was investigated by electrospray tandem mass spectrometry with the focus on the fragmentation mechanism. Results reveal new aspects of the fragmentation mechanism of modified and unmodified oligonucleotides and demonstrate the influence of the nucleobases on the decomposition of oligonucleotides. Adenine-rich oligonucleotides fragment easily resulting in abundant peaks corresponding to the DNA-typical a-B- and w-ions. On the other hand, thymine was found to have a stabilizing effect, which is reflected by the preferred formation of the w(4)-ions and the relatively low abundance of shorter w-ions upon dissociation of pentanucleotides. Data from investigation of the formation of w(4)-ions support a beta-elimination mechanism. Results obtained by investigation of oligonucleotides with an abasic site confirm this mechanism, which is independent of nucleobase loss. Experiments with methylphosphonate oligonucleotides show a remarkable change in the fragmentation pattern due to the modification. It was found that charges are located on the nucleobases and initiate the fragmentation mechanism. The stability of the oligonucleotide is reduced and no a-B-fragment ions are formed wherever there is a methylphosphonate group within the backbone. This fact also demonstrates that fragmentation is locally controlled.

Adequate design of customized cDNA macroarray for convenient multiple gene expression analysis

To establish a convenient, cost-effective, and reasonably reliable method for monitoring multiple gene expression using customized membrane-based macroarray, we constructed a cDNA macroarray with multiple probes for 13 human vascular endothelial genes and assessed the accuracy of the macroarray measurements. For each gene, two cDNA probes (450-550 bp) were designed from different regions (coding region and 3'-untranslated region [3'-UTR], respectively) on the basis of simple criteria concerning length and sequence specificity and spotted on the macroarray. In addition, unmodified oligonucleotide probes (80 mer) targeted to a unique sequence from the coding region of each gene were spotted on the same macroarray. Using this macroarray, shear stress-induced mRNA expression changes were analyzed in human coronary artery endothelial cells. Comparison of the expression ratios obtained with those measured using quantitative real-time polymerase chain reaction (PCR) as a reference method revealed that cDNA probes designed from a sequence within the coding region provided a highly accurate expression profile, whereas results obtained from oligonucleotide probes showed no correlation with real-time PCR data, which might be caused by inadequate immobilization of oligonucletotide probes on the nylon membrane. In addition, we observed that cDNA probes targeting different regions of a gene yielded different signal intensities. Most cDNA probes designed from a sequence within the coding region showed detectable signals, whereas few cDNA probes designed from 3'-UTR did.

A hexanucleotide selected for increased cellular uptake in cis contains a highly active CpG‐motif in human B cells and primary peripheral blood mononuclear cells

The relationship between immunostimulation of human B cells by cytosine-phosphate-guanosine (CpG) -containing oligonucleotides and their physical cellular uptake is of mechanistic interest and a prerequisite for rational improvements of the therapeutic potential of CpG-harbouring oligonucleotides. Here, a combinatorial approach was used to identify nucleotide sequence motifs that facilitate increased cellular uptake in mammalian cells. Oligonucleotides harbouring the selected hexanucleotide TCGTGT in cis show increased cellular uptake. This motif contains a CpG dinucleotide within a sequence context that shows a very strong CpG-specific stimulatory activity on human B cells. Here we describe the influence of concentration, length and sequence position of the unmethylated CpG dinucleotide on immunostimulation. A comparison between phosphorothioate-derivatives and unmodified TCGTGT-containing oligonucleotides strongly indicates a great CpG-specificity for the unmodified CpG-harbouring oligonucleotides but not for the phosphorothioate versions. This work describes a link between the physical cellular uptake of naked oligonucleotides harbouring the selected cellular uptake motif TCGTGT, its strong CpG-specific stimulation of human B cells and its relationship with the sequence context of CpG and its cellular uptake.

Discovery and development of anticancer aptamers

Aptamers, also termed as decoys or "chemical antibodies," represent an emerging class of therapeutics. They are short DNA or RNA oligonucleotides or peptides that assume a specific and stable three-dimensional shape in vivo, thereby providing specific tight binding to protein targets. In some cases and as opposed to antisense oligonucleotides, effects can be mediated against extracellular targets, thereby preventing a need for intracellular transportation. The first aptamer approved for use in man is a RNA-based molecule (Macugen, pegaptanib) that is administered locally (intravitreally) to treat age-related macular degeneration by targeting vascular endothelial growth factor. The most advanced aptamer in the cancer setting is AS1411, formerly known as AGRO100, which is being administered systemically in clinical trials. AS1411 is a 26-mer unmodified guanosine-rich oligonucleotide, which induces growth inhibition in vitro, and has shown activity against human tumor xenografts in vivo. The mechanism underlying its antiproliferative effects in cancer cells seems to involve initial binding to cell surface nucleolin and internalization, leading to an inhibition of DNA replication. In contrast to other unmodified oligonucleotides, AS1411 is relatively stable in serum-containing medium, probably as a result of the formation of dimers and a quartet structure. In a dose escalation phase I study in patients with advanced solid tumors, doses up to 10 mg/kg/d (using a four or seven continuous infusion regime) have been studied. Promising signs of activity have been reported (multiple cases of stable disease and one near complete response in a patient with renal cancer) in the absence of any significant adverse effects. Further trials are ongoing in renal and non-small cell lung cancers. In preclinical studies, additional aptamers have been described against several cancer targets, such as tenascin-C, the transcription factor signal transducer and activator of transcription 3, and antiapoptotic and Ku proteins.

Generation of a mouse mutant by oligonucleotide-mediated gene modification in ES cells

Oligonucleotide-mediated gene targeting is emerging as a powerful tool for the introduction of subtle gene modifications in mouse embryonic stem (ES) cells and the generation of mutant mice. However, its efficacy is strongly suppressed by DNA mismatch repair (MMR). Here we report a simple and rapid procedure for the generation of mouse mutants using transient down regulation of the central MMR protein MSH2 by RNA interference. We demonstrate that under this condition, unmodified single-stranded DNA oligonucleotides can be used to substitute single or several nucleotides. In particular, simultaneous substitution of four adjacent nucleotides was highly efficient, providing the opportunity to substitute virtually any given codon. We have used this method to create a codon substitution (N750F) in the Rb gene of mouse ES cells and show that the oligonucleotide-modified Rb allele can be transmitted through the germ line of mice.

AS1411, a Novel DNA Aptamer as a Potential Treatment of Acute Myelogenous Leukaemia (AML).

AS1411 (formerly AGRO100) is the first nucleic acid aptamer to enter oncology trials and is currently in a Phase I study in renal and lung patients. AS1411 is a 26mer unmodified phosphodiester-based guanosine rich oligonucleotide which, as a dimer, forms a serum-resistant quartet structure. It is postulated to exert anti-cancer activity by binding to cell surface nucleolin resulting in S-phase cell cycle arrest. We have previously shown that AS1411 inhibits growth of a wide range of solid tumours, including lung, renal, prostate and colorectal with EC50s of 2 to 8 μM. In this series of studies, we investigated the potential of AS1411 in the treatment of acute myelogenous leukaemia (AML). Expression of cell-surface and intracellular expression of nucleolin was assessed by indirect immunocytochemical staining. Four AML cell lines (MV4-11, KG1, HL60 and K562) and two solid tumour cell lines (A549 - lung; DU145 - prostate) were used. Permeabilised or non-permeabilised cells were either stained in situ on chamber glass slides or as cytospin preparations. Three anti-nucleolin monoclonal antibodies and the Vector immunoperoxidase system were used. Cells were qualitatively assessed for extent and intensity of staining. In the KG1, HL60 and MV4-11 cell lines, intense, homogeneous staining was seen in 100% of non-permeabilised cells, indicating high levels of surface-expressed nucleolin; in K562 cell lines, very intense, but heterogeneous surface staining was seen. In the A549 lung and DU145 prostate lines, moderate surface staining for nucleolin was seen. In the permeabilised AML and solid tumour cells, all lines showed similar levels of homogeneous, intense intracellular staining, as expected for an ubiquitous intracellular and intranuclear protein. In SRB in vitro cytotoxicity assays, AS1411 resulted in cell death with an EC50 of 0.6 to 5 μM; the MV4-11 line was most sensitive; the EC50 for solid tumour cell lines ranged between 2 and 8 μM. Since exposure of cells to AS1411 results in S-phase cell cycle arrest; we postulated that combination of AS1411 with S-phase dependent chemotherapeutic agents may result in increased cytotoxicity and synergistic activity. An early study of the combination of AS1411 with cytosine arabinoside (AraC) indicates that there is a synergistic effect between the two drugs; the data also suggest that the synergy may be schedule dependent and requires prior exposure to AS1411 before treatment with AraC. The distribution of the aptamer in the mouse was examined using tritiated-AS1411. A total dose of 158 kBq of 3H-AS1411 was given by intravenous injection to mice bearing solid human tumour xenografts and samples of tumours and normal tissue taken between 1 hour and 4 days post injection. Approximately 1.6 to 3.1% of the injected dose was measured in the bone and bone marrow 24 hours after dosing. The ratio of the amount of 3H-AS1411 in the bone marrow to that in the blood ranged between 1.3 and 3.7 throughout the 4-day sampling period. In conclusion, we have demonstrated that nucleolin appears to be intensely expressed on the surface of AML cell lines. AML cell lines are killed by treatment with AS1411 and synergistic cytotoxicity is seen when the AS1411 is combined with AraC. In addition, the bone marrow compartment is accessible to AS1411. Taken together these findings support investigating the anti-cancer effects of AS1411 in AML patients either alone or in combination with standard AraC therapy.

LNA-modified oligonucleotides effectively drive intramolecular-stable hairpin to intermolecular-duplex state

Sequence-specific hybridization of antisense and antigene agent to the target nucleic acid is an important therapeutic strategy to modulate gene expression. However, efficiency of such agents falls due to inherent intramolecular-secondary-structures present in the target that pose competition to intermolecular hybridization by complementary antisense/antigene agent. Performance of these agents can be improved by employing structurally modified complementary oligonucleotides that efficiently hybridize to the target and force it to transit from an intramolecular-structured-state to an intermolecular-duplex state. In this study, the potential of variably substituted locked nucleic acid-modified oligonucleotides (8mer) to hybridize and disrupt highly stable, secondary structure of nucleic acid has been biophysically characterized and compared with the conventionally used unmodified DNA oligonucleotides. The target here is a stem–loop hairpin oligonucleotide—a structure commonly present in most structured-nucleic acids and known to exhibit an array of biological functions. Using fluorescence-based studies and EMSA we prove that LNA-modified oligonucleotides hybridize to the target hairpin with higher binding affinity even at lower concentration and subsequently, force it to assume a duplex conformation. LNA-modified oligonucleotides may thus, prove as potential therapeutic candidates to manipulate gene expression by disruption of biologically relevant nucleic acid secondary structure.

Oligonucleotide-Modified Gold Nanoparticles for Intracellular Gene Regulation

We describe the use of gold nanoparticle-oligonucleotide complexes as intracellular gene regulation agents for the control of protein expression in cells. These oligonucleotide-modified nanoparticles have affinity constants for complementary nucleic acids that are higher than their unmodified oligonucleotide counterparts, are less susceptible to degradation by nuclease activity, exhibit greater than 99% cellular uptake, can introduce oligonucleotides at a higher effective concentration than conventional transfection agents, and are nontoxic to the cells under the conditions studied. By chemically tailoring the density of DNA bound to the surface of gold nanoparticles, we demonstrated a tunable gene knockdown.

Improved surface sensing of DNA on gas-etched porous silicon

A new gas-based etching process has been used to produce porous silicon (PSi) samples as sensor substrates. The as-etched porous material demonstrated an intense photoluminescence signal at an average wavelength of 658 nm ( σ = 1%). The photoluminescence signal, with a peak wavelength in the range 530–540 nm, was found to increase with the length and concentration of unmodified oligonucleotide probes on surfaces coated with either amino-propyl trimethoxysilane (APTMS) or glycidoxy-propyl trimethoxysilane (GPTMS). The large surface area that characterized this material significantly increased the binding of DNA probes and led to increased sensitivity in target detection. Using fluorescently-labeled DNA, the target capture for sub-micromolar detection on this novel sensor substrate was demonstrated to be 100 times higher than that obtained on non-etched silicon. In a 1 h assay, we were able to detect specifically approximately 3 fmole of a 25mer target oligonucleotide per mm 2 of substrate.

Unusual chromatographic behavior of oligonucleotide sequence isomers on two different anion exchange HPLC columns

The retention behavior of the unmodified phosphodiester oligonucleotide sequence isomers was investigated on two different anion exchange columns: Biospher GMB 1000Q (based on DEAE-modified glycidyl methacrylate) and PolyWAX LP (based on silica with a crosslinked coating of linear polyethyleneimine). There was a notable difference in retention of oligonucleotides of the same composition but differing in the position of a single base. The most pronounced difference was observed between the oligonucleotides with the variable base in the end and in the center of the sequence. The use of either acetonitrile or 2-propanol as a mobile phase organic modifier did not markedly affect the retention time patterns. Prediction of the retention times of oligonucleotides must take into account the base position as well as identity. This is the first report of such a “same composition different sequence” effect, described for the short peptides, for synthetic oligonucleotides.

Light‐Induced Formation of G‐Quadruplex DNA Secondary Structures

A little light release: Temporarily blocked (“caged”) dG nucleosides that can be incorporated into oligodeoxynucleotides are presented (see figure). Their hydrogen-bond-recognition site is inaccessible in the inactive form; this prevents, for example, the formation of secondary structures. Light can trigger the release of the unmodified oligonucleotide and thus induce correct folding.

Molecular Dynamics and Free Energy Calculations of the B and Z Forms of C8-Arylguanine Modified Oligonucleotides

Arylhydrazines found in the mushroom Agaricus bisporus have been shown to be carcinogenic. Upon metabolic activation, arylhydrazines are transformed into aryl radicals, forming 8-arylpurines, which may play a role in arylhydrazine carcinogenesis. These adducts are poorly read and inhibit chain extension but do alter the conformational preferences of oligonucleotides. We have shown that C8-phenylguanine modification of d(CGCGCG*CGCG) (G*= 8-phenylguanine) stabilizes it in the Z-DNA conformation (B/Z-DNA=1:1, 200 mM NaCl, pH 7.4). Here we have conducted molecular dynamics and free energy calculations to determine the sources(s) of these conformational affects and to predict the affect of the related C8- tolyl and C8-hydroxymethylphenyl guanine adducts on B/Z-DNA equilibrium. Force field parameters for the modified guanines were first developed using Guassian98 employing the B3LYP method and the standard 6–31G* basis set and fit to the Cornell 94 force field with RESP. Molecular dynamics simulations and free energy calculations, using the suite of programs contained in Amber 6 and 7 with the Cornell 94 force field, were used to determine the structural and thermodynamic properties of the DNA. The principal factors that drive conformation are stacking of the aryl group over the 5′-cytosine in the phenyl and tolyl modified oligonucleotides while hydrogen bonding opposes stacking in the hydroxymethylphenyl derivative. The phenyl and tolyl-modified DNA's favored the Z-DNA form as did the hydroxymethylphenyl derivative when hydrogen bonding was not present. The B-DNA conformation was preferred by the unmodified oligonucleotide and by the hydroxymethylphenyl-modified oligonucleotide when hydrogen bonding was considered. Z-DNA stability was not found to directly correlated with carcinogenicity and additional biological factors, such as recognition and repair, may also need to be considered in addition to Z-DNA formation.

Retention Behavior of Oligonucleotides on a Glycidyl Methacrylate-Based DEAE-Modified Sorbent

The retention behavior of unmodified phosphodiester mixed-sequence oligonucleotides has been investigated under different conditions on a glycidyl methacrylate-based, DEAE-modified anion-exchange Biospher GMB 1000Q column. It was verified that separation is based not only on an anion-exchange mechanism but also on secondary interactions of the bases with the stationary phase. Notably different retention was observed for oligonucleotides differing only in the position of one base in the sequence. The nature of these secondary interactions and the effect of sequence on retention is discussed. The effect on retention of the type and concentration of organic mobile phase modifier, mobile phase pH, sample molecular weight, and the bases in the sample was investigated. A linear relationship was found between retention and the molecular weight of mixed-sequence oligonucleotides with lengths from 15 to 40. Resolution of N − 1 synthetic by-products from the full-length product was also evaluated.

Oligodeoxynucleotides Containing Diastereomeric O2′,C3′‐linked Bicyclic Nucleotide Units for Functionalization of the Major Groove of Nucleic Acid Duplexes: A Summary and Novel Derivatives*

The synthesis and evaluation of a range of piperazino‐derivatized diastereomeric O2′,C3′‐linked bicyclic nucleotides are described. A new and optimized protocol is presented for the synthesis of the bicyclic scaffold on which the piperazino moiety is appended. At low salt concentration, the C2″‐S‐configured piperazino‐modified oligonucleotides display significantly enhanced hybridization affinity toward complementary DNA and RNA targets relative to the unmodified oligonucleotide control, whereas no melting transition is observed for hybrids formed with the C2″‐R‐configured piperazino‐modified oligonucleotides. Upon derivatization of the piperazino moiety with a 1‐pyrenebutanoyl group, all modified oligonucleotides display strong DNA binding and profound DNA hybridization selectivity. * Dedicated to the memory of professor Jacques H. van Boom.

Application of Molecular Dynamics Simulations to Spin-labeled Oligonucleotides

The EPR study of spin labeled macromolecules has provided insight into structural and dynamical properties of DNA, proteins, and related systems. While spin labeling has been useful, it is experimentally difficult to determine if the spin label significantly alters the conformation of the macromolecule to which it is attached. Molecular modeling has proven to be a powerful tool for studying structure and dynamics of biologically important molecules. Here, we have conducted molecular dynamics (MD) studies of spin labeled oligonucleotides (ONs) bearing a five (5sp) or six (6sp) membered ring nitroxide, and the corresponding unmodified ON using the suite of programs contained in Amber 5.0 with the Cornell et al. 94 force field (Cornell, W. D., Cieplak, P., Bayly, C. I., Gould, I. R., Merz, Jr., K. M. Ferguson, D. M., Spellmeyer, D. C., Fox, T., Caldwell, J. W., and Kollman, P. A. A Second Generation Force Field for the Simulation of Proteins and Nucleic Acids. J. Am. Chem. Soc. 117, 5179–5197 (1995)). Quantum mechanical calculations employing the B3LYP method with the standard 6–31G1 basis set using Gaussian98 were performed and, together with available crystallographic data for analogous nitroxides, new parameters for the nitrogen, oxygen, nitroxide α-carbon, and sp-hybridized carbon atoms have been developed suitable for the Cornell et al. 94 force field. MD simulations on the double-stranded (ds) spin labeled ONs, along with the corresponding unmodified analogues, have been studied over the course of 4 ns and conformational properties of all ONs are described based on the analysis of the trajectories. The spin labels were found to alter the global conformation of the ONs to which they were attached to accommodate the spin labels. The major changes include widening the major groove, decreasing helical twist, and more negative X-displacement of the base pairs. The magnitude of the effect was dependent on the specific structure of the spin label. Average and ‘most representative’ structures derived from the molecular dynamics simulations correlate with the experimental data on the spin labeled ONs.

Targeting and Regulation of the HER-2/neu Oncogene Promoter with Bis-Peptide Nucleic Acids

Antigene oligonucleotides have the potential to regulate gene expression through site-specific DNA binding. However, in vivo applications have been hindered by inefficient cellular uptake, degradation, and strand displacement. Peptide nucleic acids (PNAs) address several of these problems, as they are resistant to degradation and bind DNA with high affinity. We designed two cationic pyrimidine bis-PNAs (cpy-PNAs) to target the polypurine tract of the HER-2/neu promoter and compared them to an unmodified phosphodiester triplex-forming oligonucleotide (TFO1) and a TFO-nitrogen mustard conjugate (TFO2). PNA1 contains a + 2 charge and bound two adjacent 9-bp target sequences with high affinity and specificity, but only at low pH. PNA2 contains a +5 charge and bound one 11-bp target with high affinity up to pH 7.4, but with lower specificity. The PNA:DNA:PNA triplex formed by these cpy-bis-PNAs presented a stable barrier to DNA polymerase extension. The cpy-bis-PNAs and the TFO-alkylator conjugate prevented HER-2/neu transcription in a reporter gene assay (TFO2 = PNA1 > PNA2 >> TFO1). Both PNAs and TFOs were effective at binding the target sequence in naked genomic DNA, but only the TFO-alkylator (TFO2) and the more cationic PNA (PNA2) were detected at the endogenous HER-2/neu promoter in permeabilized cells. This work demonstrates the potential for preventing HER-2/neu gene expression with cpy-bis-PNAs in tumor cells.

Structure of a High Fidelity DNA Polymerase Bound to a Benzo[a]pyrene Adduct That Blocks Replication

Of the carcinogens to which humans are most frequently exposed, the polycyclic aromatic hydrocarbon benzo[a]pyrene (BP) is one of the most ubiquitous. BP is a byproduct of grilled foods and tobacco and fuel combustion and has long been linked to various human cancers, particularly lung and skin. BP is metabolized to diol epoxides that covalently modify DNA bases to form bulky adducts that block DNA synthesis by replicative or high fidelity DNA polymerases. Here we present the structure of a high fidelity polymerase from a thermostable strain of Bacillus stearothermophilus (Bacillus fragment) bound to the most common BP-derived N2-guanine adduct base-paired with cytosine. The BP adduct adopts a conformation that places the polycyclic BP moiety in the nascent DNA minor groove and is the first structure of a minor groove adduct bound to a polymerase. Orientation of the BP moiety into the nascent DNA minor groove results in extensive disruption to the interactions between the adducted DNA duplex and the polymerase. The disruptions revealed by the structure of Bacillus fragment bound to a BP adduct provide a molecular basis for rationalizing the potent blocking effect on replication exerted by BP adducts.

Hybridization and Melting Behavior of Peptide Nucleic Acid (PNA) Oligonucleotide Chimeras Conjugated to Gold Nanoparticles

AbstractPeptide nucleic acids (PNA) and PNA–DNA chimeras carrying thiol groups were used for surface functionalization of Au nanoparticles. Conjugation of PNA to citrate‐stabilized Au nanoparticles destabilized the nanoparticles causing them to precipitate. Addition of a tail of glutamic acid to the PNA prevented destabilization of the nanoparticles but resulted in loss of interaction with complementary sequences. Importantly, PNA–DNA chimeras gave stable conjugates with Au nanoparticles. The hybridization and melting properties of complexes formed from chimera–nanoparticle conjugates and oligonucleotide–nanoparticle conjugates are described for the first time. Similar to oligonucleotide–nanoparticle conjugates, conjugates with PNA–DNA chimeras gave sharper and more‐defined melting profiles than those obtained with unmodified oligonucleotides. In addition, mismatch discrimination was found to be more efficient than with unmodified oligonucleotides.