Tullia Tedeschi

Insights into peptide nucleic acid (PNA) structural features: The crystal structure of a D-lysine-based chiral PNA-DNA duplex

Peptide nucleic acids (PNAs) are oligonucleotide analogues in which the sugar-phosphate backbone has been replaced by a pseudopeptide skeleton.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-Å resolution by means of a single-wavelength anomalous diffraction experiment on a brominated derivative. Thed-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.

Chiral introduction of positive charges to PNA for double-duplex invasion to versatile sequences

Invasion of two PNA strands to double-stranded DNA is one of the most promising methods to recognize a predetermined site in double-stranded DNA (PNA = peptide nucleic acid). In order to facilitate this ‘double-duplex invasion’, a new type of PNA was prepared by using chiral PNA monomers in which a nucleobase was bound to the α-nitrogen of N-(2-aminoethyl)-d-lysine. These positively charged monomer units, introduced to defined positions in Nielsens PNAs (poly[N-(2-aminoethyl)glycine] derivatives), promoted the invasion without impairing mismatch-recognizing activity. When pseudo-complementary nucleobases 2,6-diaminopurine and 2-thiouracil were bound to N-(2-aminoethyl)-d-lysine, the invasion successfully occurred even at highly G–C-rich regions [e.g. (G/C)7(A/T)3 and (G/C)8(A/T)2] which were otherwise hardly targeted. Thus, the scope of sequences available as the target site has been greatly expanded. In contrast with the promotion by the chiral PNA monomers derived from N-(2-aminoethyl)-d-lysine, their l-isomers hardly invaded, showing crucial importance of the d-chirality. The promotion of double-duplex invasion by the chiral (d) PNA monomer units was ascribed to both destabilization of PNA/PNA duplex and stabilization of PNA/DNA duplexes.

Peptide Nucleic Acids with a Structurally Biased Backbone. Updated Review and Emerging Challenges

Peptide nucleic acids (PNAs) are polyamidic oligonucleotide analogues which have been described for the first time almost twenty years ago and were immediately found to be excellent tool in binding DNA and RNA for diagnostics and gene regulation. Their use as therapeutic agents have been proposed since early studies and recent advancements in cellular delivery systems and in the so called anti-gene strategy make them good candidates for drug development. The search for new chemical modification of PNAs is a very active field of research and new structures are continuously proposed. This review focuses on the modification of the PNA backbone, and their possible use in medicinal chemistry with an update of this topics in view of emerging new trends and opening of new possibilities In particular two classes of structurally biased PNAs are described in details: i) PNAs with acyclic structures and their helical preference, which is regulated by stereochemistry and ii) cyclic PNAs with preorganized structures, whose performances depend both on stereochemistry and on conformational constraints. The properties of these compounds are discussed in terms of affinity for nucleic acids, and several examples of their use in cellular or animal systems are presented , with exciting new fields of research such as microRNA (miR) targeting and gene repair.

Modulation of the biological activity of microRNA-210 with peptide nucleic acids (PNAs).

Herein we describe the activity of a peptide nucleic acid (PNA) that targets microRNA‐210 (miR‐210), which is associated with hypoxia and is modulated during erythroid differentiation. PNAs directed against miR‐210 were designed to bind with high affinity to the target RNA strand and to undergo efficient uptake in target cells. A polyarginine–PNA conjugate directed against miR‐210 (Rpep‐PNA‐a210) showed both very high affinity for RNA and efficient uptake into target cells without the need for transfection reagents. An unmodified PNA of the same sequence displayed the ability to bind RNA, but cellular uptake was very poor. Consistent with this, only Rpep‐PNA‐a210 strongly inhibited miR‐210 activity, as evaluated by assays on undifferentiated K562 cells and on cells treated with mithramycin, which was found to induce erythroid differentiation and miR‐210 overexpression. Targeting miR‐210 by Rpep‐PNA‐a210 resulted in: 1) a decrease in miR‐210 levels as measured by RT‐PCR, 2) up‐regulation of raptor mRNA, 3) a decrease in γ‐globin mRNA, and 4) decreased expression of differentiated functions (i.e., proportion of benzidine‐positive cells, content of embryo‐fetal hemoglobins). The efficient delivery of anti‐miR PNAs through a suitable peptide carrier (Rpep‐PNA‐a210) leads to the inhibition of miR‐210 activity, altering the expression of miR‐210‐regulated erythroid functions.

Pectin content and composition from different food waste streams.

In the present paper, 26 food waste streams were selected according to their exploitation potential and investigated in terms of pectin content. The isolated pectin, subdivided into calcium bound and alkaline extractable pectin, was fully characterized in terms of uronic acid and other sugar composition, methylation and acetylation degree. It was shown that many waste streams can be a valuable source of pectin, but also that pectin structures present a huge structural diversity, resulting in a broad range of pectin structures. These can have different physicochemical and biological properties, which are useful in a wide range of applications. Even if the data could not cover all the possible batch by batch and country variabilities, to date this represents the most complete pectin characterization from food waste streams ever reported in the literature with a homogeneous methodology.

Peptide Nucleic Acids with a Structurally Biased Backbone: Effects of Conformational Constraints and Stereochemistry

Peptide nucleic acids (PNAs) are polyamidic oligonucleotide analogs which have been described for the first time fifteen years ago and were immediately found to be excellent tools in binding DNA and RNA for diagnostics and gene regulation. Their use as therapeutic agents have been proposed since early studies and recent advancements in cellular delivery systems, and in the so called anti-gene strategy, makes them good candidates for drug development. The search for new chemical modification of PNAs is a very active field of research and new structures are continuously proposed. This review focuses on the recent advancements obtained by the modification of the PNA backbone, and their possible use in medicinal chemistry. In particular two classes of structurally biased PNAs are described in details: i) PNAs with acyclic structures and their helical preference, which is regulated by stereochemistry and ii) cyclic PNAs with preorganized structures, whose performances depend both on stereochemistry and on conformational constraints. The properties of these compounds are discussed in terms of affinity for nucleic acids, and several recent examples of their use in cellular or animal systems are presented.

Racemization of chiral PNAs during solid-phase synthesis: effect of the coupling conditions on enantiomeric purity

Abstract Chiral peptide nucleic acid (PNA) monomers based on the amino acids Ala, Phe and Lys were synthesized, and their enantiomeric purity was checked either by RP-HPLC after reaction with l -ValOMe or by GC–MS using a Chirasil-Val column after hydrolysis and conversion of PNAs to the corresponding piperazin-2-ones. A model coupling reaction of these monomers with ValOMe was carried out under various conditions in order to evaluate the effect of synthetic parameters (coupling agent, base, preactivation time) on epimerization. The enantiomeric purity of the products decreased in the order: DEPBT>TDBTU>HBTU>HATU. The use of sym -collidine (TMP) as a base produced higher racemization than with DIEA. PNAs containing one or three chiral monomers were subsequently synthesized with different coupling protocols, and the results were found to be consistent with those obtained in solution. High enantiomeric purity was obtained using a DIC/HOBt coupling protocol. A rationale for the observed effects is proposed based on NMR studies.

Cellular Uptakes, Biostabilities and Anti‐miR‐210 Activities of Chiral Arginine‐PNAs in Leukaemic K562 Cells

A series of 18‐mer peptide nucleic acids (PNAs) targeted against micro‐RNA miR‐210 was synthesised and tested in a cellular system. Unmodified PNAs, R8‐conjugated PNAs and modified PNAs containing eight arginine residues on the backbone, either as C2‐modified (R) or C5‐modified (S) monomers, all with the same sequence, were compared. Two different models were used for the modified PNAs: one with alternated chiral and achiral monomers and one with a stretch of chiral monomers at the N terminus. The melting temperatures of these derivatives were found to be extremely high and 5 M urea was used to assess differences between the different structures. FACS analysis and qRT‐PCR on K562 chronic myelogenous leukaemic cells indicated that arginine‐conjugated and backbone‐modified PNAs display good cellular uptake, with best performances for the C2‐modified series. Resistance to enzymatic degradation was found to be higher for the backbone‐modified PNAs, thus enhancing the advantage of using these derivatives rather than conjugated PNAs in the cells in serum, and this effect is magnified in the presence of peptidases such as trypsin. Inhibition of miR‐210 activity led to changes in the erythroid differentiation pathway, which were more evident in mithramycin‐treated cells. Interestingly, the anti‐miR activities differed with use of different PNAs, thus suggesting a role of the substituents not only in the cellular uptake, but also in the mechanism of miR recognition and inactivation. This is the first report relating to the use of backbone‐modified PNAs as anti‐miR agents. The results clearly indicate that backbone‐modified PNAs are good candidates for the development of very efficient drugs based on anti‐miR activity, due to their enhanced bioavailabilities, and that overall anti‐miR performance is a combination of cellular uptake and RNA binding.

miRNA therapeutics: delivery and biological activity of peptide nucleic acids targeting miRNAs.

Peptide nucleic acids (PNAs) are DNA/RNA mimics extensively used for pharmacological regulation of gene expression in a variety of cellular and molecular systems, and they have been described as excellent candidates for antisense and antigene therapies. At present, very few data are available on the use of PNAs as molecules targeting miRNAs. miRNAs are a family of small nc RNAs that regulate gene expression by sequence-selective targeting of mRNAs, leading to a translational repression or mRNA degradation to the control of highly regulated biological functions, such as differentiation, cell cycle and apoptosis. The aim of this article is to present the state-of-the-art concerning the possible use of PNAs to target miRNAs and modify their biological metabolism within the cells. The results present in the literature allow to propose PNA-based molecules as very promising reagents to modulate the biological activity of miRNAs. In consideration of the involvement of miRNAs in human pathologies, PNA-mediated targeting of miRNAs has been proposed as a potential novel therapeutic approach.

Arginine-based PNA microarrays for APOE genotyping

Four modified PNAs containing one chiral monomer bearing two arginine-derived side chains, with the correct configuration for specific and stable DNA binding, were synthesized, complementary to two DNA tracts in the APOE gene containing SNPs related to the insurgence of Alzheimers disease. PNA binding performances were first tested in solution against complementary and mismatched oligonucleotides by measuring melting temperatures, and showed high specificity in SNP recognition. In order to set up a new diagnostic platform for APOE genotyping, PNA microarrays were then developed with the synthesized modified PNAs. PNA probe deposition protocols on microarrays were optimized in order to minimize cross-contamination due to carry over. The microarrays obtained by arginine-based PNA deposition were incubated with complementary and mismatched oligonucleotides, showing excellent mismatch recognition on the microarray platform. The specificity of the microarrays was finally tested with oligonucleotide mixtures simulating the real genotype profiles. Six different hybridisation patterns related to six different genotypes in the APOE gene were found to be clearly distinct in microarray experiments, demonstrating the potential of this approach for highly specific genetic analysis.