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Organosilica nanoparticles that contain responsive organic building blocks as constitutive components of the silica network offer promising opportunities for the development of innovative drug formulations, biomolecule delivery, and diagnostic tools. However, the synthetic challenges required to introduce dynamic and multifunctional building blocks have hindered the realization of biomimicking nanoparticles. In this study, capitalizing on our previous research on responsive nucleic acid-based organosilica nanoparticles, we combine the supramolecular programmability of nucleic acid (NA) interactions with sol-gel chemistry. This approach allows us to create dynamic supramolecular bridging units of nucleic acids in a silica-based scaffold. Two peptide nucleic acid-based monoalkoxysilane derivatives, which self-assemble into a supramolecular bis-alkoxysilane through direct base pairing, were chosen as the noncovalent units inserted into the silica network. In addition, a bridging functional NA aptamer leads to the specific recognition of ATP molecules. In a one-step bottom-up approach, the resulting supramolecular building blocks can be used to prepare responsive organosilica nanoparticles. The supramolecular Watson-Crick-Franklin interactions of the organosilica nanoparticles result in a programmable response to external physical (i.e., temperature) and biological (i.e., DNA and ATP) inputs and thus pave the way for the rational design of multifunctional silica materials with application from drug delivery to theranostics.

The development of smart nanoparticles (NPs) that encode responsive features in the structural framework promises to extend the applications of NP-based drugs, vaccines, and diagnostic tools. New nanocarriers would ideally consist of a minimal number of biocompatible components and exhibit multiresponsive behavior to specific biomolecules, but progress is limited by the difficulty of synthesizing suitable building blocks. Through a nature-inspired approach that combines the programmability of nucleic acid interactions and sol-gel chemistry, we report the incorporation of synthetic nucleic acids and analogs, as constitutive components, into organosilica NPs. We prepared different nanomaterials containing single-stranded nucleic acids that are covalently embedded in the silica network. Through the incorporation of functional nucleic acids into the organosilica framework, the particles respond to various biological, physical, and chemical inputs, resulting in detectable physicochemical changes. The one-step bottom-up approach used to prepare organosilica NPs provides multifunctional systems that combine the tunability of oligonucleotides with the stiffness, low cost, and biocompatibility of silica for different applications ranging from drug delivery to sensing.

The understanding of the interaction of chiral species with DNA or RNA is very important for the development of new tools in biology and of new drugs. Several cases in which chirality is a crucial point in determining the DNA binding mode are reviewed and discussed, with the aim of illustrating how chirality can be considered as a tool for improving the understanding of mechanisms and the effectiveness of nucleic acid recognition. The review is divided into two parts: the former describes examples of chiral species interacting with DNA: intercalators, metal complexes, and groove binders; the latter part is dedicated to chirality in DNA analogs, with discussion of phosphate stereochemistry and chirality of ribose substitutes, in particular of peptide nucleic acids (PNAs) for which a number of works have been published recently dealing with the effect of chirality in DNA recognition. The discussion is intended to show how enantiomeric recognition originates at the molecular level, by exploiting the enormous progresses recently achieved in the field of structural characterization of complexes formed by nucleic acid with their ligands by crystallographic and spectroscopic methods. Examples of application of the DNA binding molecules described and the role of chirality in DNA recognition relevant for biotechnology or medicinal chemistry are reported., ((c) 2007 Wiley-Liss, Inc.)

Peptide nucleic acids are DNA mimics able to form duplexes with complementary DNA or RNA strands of remarkable affinity and selectivity. Oligopyrimidine PNA can displace one strand of dsDNA by forming PNA(2):DNA triplexes of very high stability. Many PNA analogs have been described in recent years, in particular, chiral PNA analogs. In the present article the results obtained recently using PNA derived from N-aminoethylamino acids 7 are illustrated. In particular, the dependence of optical purity on synthetic methodologies and a rationale for the observed effects of chirality on DNA binding ability is proposed. Chirality as a tool for improving sequence selectivity is also described. PNA analogs derived from D- or L-ornithine 8 were also found to be subjected to epimerization during solid phase synthesis. Modification of the coupling conditions or the use of a submonomeric strategy greatly reduced epimerization. The optically pure oligothymine PNAs 8 were found to bind to RNA by forming triplexes of unusual CD spectra. The melting curves of these adducts presented two transitions, suggesting a conformational change followed by melting at high temperature., (Copyright 2002 Wiley-Liss, Inc.)

Simple Summary: Starting from a list of nine miRNAs found to be dysregulated in the bloodstream of early-stage colon cancer (CRC) patients, the biological effects of the targeting of the most relevant up-regulated miRNAs with anti-miRNA peptide nucleic acids (PNAs) were verified. The down-regulation of these target miRNAs with PNAs is associated with apoptosis induction in CRC cellular models. Apoptosis induction, which is one of the most important anticancer mechanisms, reaches very high levels when anti-miRNAs PNAs are combined with other pro-apoptotic compounds such as sulforaphane. Liquid biopsy has dramatically changed cancer management in the last decade; however, despite the huge number of miRNA signatures available for diagnostic or prognostic purposes, it is still unclear if dysregulated miRNAs in the bloodstream could be used to develop miRNA-based therapeutic approaches. In one author's previous work, nine miRNAs were found to be dysregulated in early-stage colon cancer (CRC) patients by NGS analysis followed by RT-dd-PCR validation. In the present study, the biological effects of the targeting of the most relevant dysregulated miRNAs with anti-miRNA peptide nucleic acids (PNAs) were verified, and their anticancer activity in terms of apoptosis induction was evaluated. Our data demonstrate that targeting bloodstream up-regulated miRNAs using anti-miRNA PNAs leads to the down-regulation of target miRNAs associated with inhibition of the activation of the pro-apoptotic pathway in CRC cellular models. Moreover, very high percentages of apoptotic cells were found when the anti-miRNA PNAs were associated with other pro-apoptotic agents, such as sulforaphane (SFN). The presented data sustain the idea that the targeting of miRNAs up-regulated in the bloodstream with a known role in tumor pathology might be a tool for the design of protocols for anti-tumor therapy based on miRNA-targeting molecules. [ABSTRACT FROM AUTHOR]

Practical laboratory classes teaching molecular pharmacology approaches employed in the development of therapeutic strategies are of great interest for students of courses in Biotechnology, Applied Biology, Pharmaceutic and Technology Chemistry, Translational Oncology. Unfortunately, in most cases the technology to be transferred to learning students is complex and requires multi-step approaches. In this respect, simple and straightforward experimental protocols might be of great interest. This study was aimed at presenting a laboratory exercise focusing (a) on a very challenging therapeutic strategy, i.e. microRNA therapeutics, and (b) on the employment of biomolecules of great interest in applied biology and pharmacology, i.e. peptide nucleic acids (PNAs). The aims of the practical laboratory were to determine: (a) the possible PNA-mediated arrest in RT-qPCR, to be eventually used to demonstrate PNA targeting of selected miRNAs; (b) the possible lack of activity on mutated PNA sequences; (c) the effects (if any) on the amplification of other unrelated miRNA sequences. The results which can be obtained support the following conclusions: PNA-mediated arrest in RT-qPCR can be analyzed in a easy way; mutated PNA sequences are completely inactive; the effects of the employed PNAs are specific and no inhibitory effect occurs on other unrelated miRNA sequences. This activity is simple (cell culture, RNA extraction, RT-qPCR are all well-established technologies), fast (starting from isolated and characterized RNA, few hours are just necessary), highly reproducible (therefore easily employed by even untrained students). On the other hand, these laboratory lessons require some facilities, the most critical being the availability of instruments for PCR. While this might be a problem in the case these instruments are not available, we would like to underline that determination of the presence or of a lack of amplified product can be also obtained using standard analytical approaches based on agarose gel electrophoresis. [ABSTRACT FROM AUTHOR]

Abstract: Technologies today available for the DNA detection rely on a combination of labeled probes hybridized to target sequences which are amplified by polymerase chain reaction (PCR). Direct detection methods that eliminate the requirement for both PCR and labeling steps could afford faster, cheaper and simpler devices for the analysis of small amounts of unamplified DNA. In this work we describe the results obtained in the ultrasensitive detection of non-amplified genomic DNA. We analyzed certified reference materials containing different amounts of genetically modified DNA by using a detection method which combines the nanoparticle-enhanced surface plasmon resonance imaging (SPRI) biosensing to the peptide nucleic acids (PNAs) improved selectivity and sensitivity in targeting complementary DNA sequences. The method allowed us to obtain a 41zM sensitivity in targeting genomic DNA even in the presence of a large excess of non-complementary DNA. [Copyright &y& Elsevier]

Peptide nucleic acids (PNAs) are mimics of oligonucleotides containing a neutral peptidelike backbone and are able to bind complementary DNA targets with high affinity and selectivity. In order to investigate the effect of the ionic strength of the buffer solution, hybridization experiments with PNAs as (catcher) probes and DNAs as target oligonucleotides were performed in different salt solutions. Surface plasmon field-enhanced fluorescence spectroscopy was employed for real-time monitoring of DNA hybridizations to surface bound PNA. Probes with three different strand lengths were immobilized by self-assembly on the sensor surface. By introducing Cy5-labeled DNA targets the affinity constants, KA=kon (association)/koff (dissociation), were determined for fully complementary (MM0) as well as for single base mismatched (MM1) duplexes. Furthermore, the thermal stability of each duplex was determined by measuring melting curves in solution which was then compared to the kinetic and affinity parameters determined for the surface hybridization reactions. The results indicate that ions do not play a significant role for the PNA/DNA hybridization kinetics at surfaces. However, changes in the configuration of the PNA/DNA duplex due to the ionic strength variations influence the fluorescence yield drastically. [ABSTRACT FROM AUTHOR]

Peptide nucleic acid (PNA), a polyamide DNA mimic, has inspired the development of a variety of hybridization-based methods for the detection, quantification, purification, and characterization of nucleic acids owing to the stability of the PNA/DNA duplex. In this work, PNA probes complementary to a specific sequence of Roundup Ready® soybean were immobilized onto a sensor surface via a self-assembled matrix employing streptavidin/biotin binding. The specific hybridization of PNA and DNA has been monitored by applying the chromophore-labeled DNA target oligonucleotides to the PNA modified Au sensor surface in real time using surface plasmon field-enhanced fluorescence spectroscopy. The authors demonstrate three kinds of experiments called global, titration, and kinetic analyses for the determination of rate constants for the association (kon) and dissociation processes (koff), and the affinity constant (KA) of the PNA/DNA duplex formation by fitting the data to a simple Langmuir model. Discrimination of a single base mismatched DNA (15mer) target on a 15mer PNA probe was documented, with a difference of the affinity constant of two orders of magnitude. Finally, the affinity constant for the hybridization of a long polymerase chain reaction product (169mer) obtained by amplification of DNA extracted from genetically modified soybean reference material has been determined by a kinetic-titration analysis. The results show the influence of a Coulomb barrier at high target surface coverage even for the hybridization to PNA at low ionic strength. [ABSTRACT FROM AUTHOR]

We have shown previously that a T10 peptide nucleic acid (PNA) bound to the transcriptional terminator of a Saccharomyces cerevisiae tDNAIle(TAT) gene arrests elongating yeast RNA polymerase (pal) III at a position that precedes by 20 bp the upstream end of the PNA roadblock (Dieci, G., Corradini, R., Sforza, S., Marchelli, R., and Ottonello, S. (2001) J. Biol. Chem. 276, 5720–5725). Here, a PNA-binding cassette was placed at various distances downstream of a functional tDNAIle transcriptional terminator (T6) that is not bound by the T10 PNA, and the effect of the PNA roadblock on RNA 3′-end formation, transcript release, and transcription reinitiation was examined. With a PNA roadblock placed as close as 5 hp downstream of the T6 terminator, pol III could still reach the termination site and complete pre-tRNA synthesis, implying that the catalytic site-to-front edge (C-F) distance of the polymerase can shorten by >10 bp upon recognition of the terminator element. In addition, transcripts synthesized by a PNA-roadblocked terminating pal III were found to be released from transcription complexes. Interestingly, however, the same roadblock dramatically reduced the rate of transcription reinitiation. Also, when placed 5 bp downstream of a mutationally inactivated terminator element (T3GT2), the PNA roadblock restored transcription termination, thus indicating that the inactivated terminator is compromised in its ability to cause pol III pausing, but can still induce C-F distance shortening and transcript release. The latter two activities were found to be further impaired in variants of the inactivated terminator bearing fewer than three consecutive T residues (T2G2T2 and TG2TGT). The data indicate that RNA polymerase pausing, C-F distance shortening, and transcript release are functionally distinguishable features of the termination process and point to the RNA release propensity of pol III as a major determinant of its remarkably high termination efficiency. [ABSTRACT FROM AUTHOR]

Peptide nucleic acids (PNAs) are oligonucleotide analogues in which the sugar-phosphate backbone has been replaced by a pseudopeptide skeleton. They bind DNA and RNA with high specificity and selectivity, leading to PNA-RNA and PNA-DNA hybrids more stable than the corresponding nucleic acid complexes. The binding affinity and selectivity of PNAs for nucleic acids can be modified by the introduction of stereogenic centers (such as D-Lys-based units) into the PNA backbone. To investigate the structural features of chiral PNAs, the structure of a PNA decamer containing three D-Lys-based monomers (namely H-GpnTpnApnGpnAdlTdlCdlApnCpnTpn-NH2, in which pn represents a pseudopeptide link and dl represents a D-Lys analogue) hybridized with its complementary antiparallel DNA has been solved at a 1.66-A resolution by means of a single-wavelength anomalous diffraction experiment on a brominated derivative. The D-Lys-based chiral PNA-DNA (LPD) heteroduplex adopts the so-called P-helix conformation. From the substantial similarity between the PNA conformation in LPD and the conformations observed in other PNA structures, it can be concluded that PNAs possess intrinsic conformational preferences for the P-helix, and that their flexibility is rather restricted. The conformational rigidity of PNAs is enhanced by the presence of the chiral centers, limiting the ability of PNA strands to adopt other conformations and, ultimately, increasing the selectivity in molecular recognition. [ABSTRACT FROM AUTHOR]

A 10-mer duplex formed between a PNA containing a 'chiral box' of three adjacent D-Lys-based monomers and its complementary DNA strand has been crystallized for the first time. Crystals have been obtained using PEG 8000 as precipitant and cacodylate at pH 6.3 as buffer. The crystals belong to the space group P31 or to its enantiomorph P32, with unit-cell parameters a = b = 35.00, c = 35.91 Å. A complete data set has been collected at the synchrotron source Elettra in Trieste to 1.85 Å resolution, using a single frozen crystal. [ABSTRACT FROM AUTHOR]

A new amperometric sandwich-format genosensor has been implemented on single-walled carbon nanotubes screen printed electrodes (SWCNT-SPEs) and compared in terms of performance with analogous genoassays developed using the same methodology on non-nanostructured glassy carbon platforms (GC-SPE). The working principle of the genosensors is based on the covalent immobilization of Peptide Nucleic Acid (PNA) capture probes (CP) on the electrode surface, carried out through the carboxylic functions present on SWCNT-SPEs (carboxylated SWCNT) or electrochemically induced on GC-SPEs. The sequence of the CP was complementary to a 20-mer portion of the target DNA; a second biotin-tagged PNA signalling probe (SP), with sequence complementary to a different contiguous portion of the target DNA, was used to obtain a sandwich hybrid with an Alkaline Phosphatase-streptavidin conjugate (ALP-Strp). Comparison of the responses obtained from the SWCNT-SPEs with those produced from the non-nanostructured substrates evidenced the remarkable enhancement effect given by the nanostructured electrode platforms, achieved both in terms of loading capability of PNA probes and amplification of the electron transfer phenomena exploited for the signal transduction, giving rise to more than four-fold higher sensitivity when using SWCNT-SPEs. The nanostructured substrate allowed to reach limit of detection (LOD) of 71 pM and limit of quantitation (LOQ) of 256 pM, while the corresponding values obtained with GC-SPEs were 430 pM and 1.43 nM, respectively. [ABSTRACT FROM AUTHOR]

Abstract: N γ-Carboxyalkyl modified peptide nucleic acids (PNAs), containing the four canonical nucleobases, were prepared via solid-phase oligomerization. The inserted peptoid monomers 1 and 2 were constructed through simple synthetic procedures, utilizing appropriate glycidol and iodoalkyl electrophiles. Thermal denaturation studies, performed with complementary antiparallel DNA strands, demonstrated that the length of the N γ-side chain strongly influences the modified PNAs hybridization properties. Moreover, multiple negative charges on the oligoamide backbone, when present on γ-nitrogen C6 side chains proved to be beneficial for the oligomers’ water solubility and DNA hybridization specificity. [Copyright &y& Elsevier]

Abstract: Lys-based ‘chiral box’ Peptide Nucleic Acids (PNAs with three adjacent 2D-Lys-based chiral monomers) have shown unsurpassed specificity in DNA recognition. In this Letter, the binding performances of arginine-based chiral PNAs were evaluated for PNAs containing in the middle part of the strand either a 2D,5L-Arg monomer or three adjacent 2D-; 2D,5L-; 5L-Arg monomers (‘Extended Chiral Box’), a combination never studied before. The binding performances of the PNAs were studied by evaluating the melting temperatures of fullmatch and mismatch PNA–DNA and PNA–RNA hybrids and by studying their structure by circular dichroism (CD). The data indicated that the arginine side chains inserted in the PNA structure are perfectly equivalent to lysine side chains as far as oligonucleotide recognition is concerned. The insertion of an ‘Extended Chiral Box’ into PNA differently influences the binding properties to DNA and RNA: the additional side chains had no observable effect on binding affinity and selectivity toward DNA, whereas, seemed to slightly disturb the binding affinity to RNA but at the same time highly enhancing the recognition selectivity. [ABSTRACT FROM AUTHOR]