Polyamide Nucleic Acid Research Articles

Translational Inhibition of CTX-M Extended Spectrum β-Lactamase in Clinical Strains of Escherichia coli by Synthetic Antisense Oligonucleotides Partially Restores Sensitivity to Cefotaxime.

Synthetic antisense oligomers are DNA mimics that can specifically inhibit gene expression at the translational level by ribosomal steric hindrance. They bind to their mRNA targets by Watson-Crick base pairing and are resistant to degradation by both nucleases and proteases. A 25-mer phosphorodiamidate morpholino oligomer (PMO) and a 13-mer polyamide (peptide) nucleic acid (PNA) were designed to target mRNA (positions -4 to +21, and –17 to –5, respectively) close to the translational initiation site of the extended-spectrum β-lactamase resistance genes of CTX-M group 1. These antisense oligonucleotides were found to inhibit β-lactamase activity by up to 96% in a cell-free translation-transcription coupled system using an expression vector carrying a blaCTX-M-15 gene cloned from a clinical isolate. Despite evidence for up-regulation of CTX-M gene expression, they were both found to significantly restore sensitivity to cefotaxime (CTX) in E. coli AS19, an atypical cell wall permeable mutant, in a dose dependant manner (0-40 nM). The PMO and PNA were covalently bound to the cell penetrating peptide (CPP; (KFF)3K) and both significantly (P < 0.05) increased sensitivity to CTX in a dose dependent manner (0-40 nM) in field and clinical isolates harboring CTX-M group 1 β-lactamases. Antisense oligonucleotides targeted to the translational initiation site and Shine-Dalgarno region of blaCTX-M-15 inhibited gene expression, and when conjugated to a cell penetrating delivery vehicle, partially restored antibiotic sensitivity to both field and clinical isolates.

Polyanionic Carboxyethyl Peptide Nucleic Acids (ce-PNAs): Synthesis and DNA Binding

New polyanionic modifications of polyamide nucleic acid mimics were obtained. Thymine decamers were synthesized from respective chiral α- and γ-monomers, and their enantiomeric purity was assessed. Here, we present the decamer synthesis, purification and characterization by MALDI-TOF mass spectrometry and an investigation of the hybridization properties of the decamers. We show that the modified γ-S-carboxyethyl-T10 PNA forms a stable triplex with polyadenine DNA.

Peculiarities of solid-phase synthesis of negatively charged chiral polyamides as nucleic acid mimics

Homopyrimidine polyamide nucleic acid mimics based on L-glutamic acid were prepared by solid-phase synthesis. The effect of the conditions of condensation and cleavage reactions with the use of polymeric support on the yield of the target sequences was studied. Possible ways of formation of by-products were revealed by mass spectrometry.

ChemInform Abstract: Fmoc/Acyl Protecting Groups in the Synthesis of Polyamide (Peptide) Nucleic Acid Monomers.

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Mechanism of RNA Cleavage Catalyzed by Sequence Specific Polyamide Nucleic Acid-Neamine Conjugate

In earlier studies, we found that a conjugate of neamine-polyamide nucleic acid targeting transactivation response element of HIV-1 RNA genome (HIV-1 TAR) displayed anti-HIV-1 activity and sequence-specific cleavage of the target RNA in vitro. Here we show that both the position of conjugation of polyamide nucleic acid (PNA) on neamine and the length of the spacer are critical parameters for conferring cleavage activity to the conjugate. The conjugation of PNA via a spacer incorporating 11 atoms to the 5-position of ring I of the neamine core conferred sequence-specific RNA cleavage activity on the conjugate, while conjugation to the 4'-position of ring II abolished this activity. Similarly, 5-neamine PNA complementary to TAR sequence of HIV-1 genome (PNA(TAR)) conjugates having either a 23-atom spacer or a bulky dansyl group between PNA and the neamine core also resulted in complete loss of cleavage activity. Based on these observations, we propose a mechanism for the observed RNA cleavage catalyzed by the conjugate involving unprotonated and protonated amino groups at the 3-position of ring I and the 6'-position of ring II of the neamine core, respectively.

Anti-HIV-1 activity of anti-TAR polyamide nucleic acid conjugated with various membrane transducing peptides

The transactivator responsive region (TAR) present in the 5′-NTR of the HIV-1 genome represents a potential target for antiretroviral intervention and a model system for the development of specific inhibitors of RNA–protein interaction. Earlier, we have shown that an anti-TAR polyamide nucleotide analog (PNATAR) conjugated to a membrane transducing (MTD) peptide, transportan, is efficiently taken up by the cells and displays potent antiviral and virucidal activity [B. Chaubey, S. Tripathi, S. Ganguly, D. Harris, R. A. Casale and V. N. Pandey (2005) Virology, 331, 418–428]. In the present communication, we have conjugated five different MTD peptides, penetratin, tat peptide, transportan-27, and two of its truncated derivatives, transportan-21 and transportan-22, to a 16mer PNA targeted to the TAR region of the HIV-1 genome. The individual conjugates were examined for their uptake efficiency as judged by FACScan analysis, uptake kinetics using radiolabeled conjugate, virucidal activity and antiviral efficacy assessed by inhibition of HIV-1 infection/replication. While FACScan analysis revealed concentration-dependent cellular uptake of all the PNATAR–peptide conjugates where uptake of the PNATAR–penetratin conjugate was most efficient as >90% MTD was observed within 1 min at a concentration of 200 nM. The conjugates with penetratin, transportan-21 and tat-peptides were most effective as an anti-HIV virucidal agents with IC50 values in the range of 28–37 nM while IC50 for inhibition of HIV-1 replication was lowest with PNATAR–transportan-27 (0.4 μM) followed by PNATAR–tat (0.72 μM) and PNATAR–penetratin (0.8 μM). These results indicate that anti-HIV-1 PNA conjugated with MTD peptides are not only inhibitory to HIV-1 replication in vitro but are also potent virucidal agents which render HIV-1 virions non-infectious upon brief exposure.

Destabilization of tRNA3(Lys) from the primer-binding site of HIV-1 genome by anti-A loop polyamide nucleotide analog.

Initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by extension of the cellular tRNA3(Lys) which anneals to the primer-binding site (PBS) on the 5' non-translated region of the viral RNA genome. The A-rich sequence (A-loop) upstream of the PBS interacts with the anticodon loop of tRNA3(Lys) and has been proposed to be essential for conferring specificity to tRNA3(Lys) for priming the initiation of HIV-1 reverse transcription. We observed that polyamide nucleic acid targeted to the A-loop sequence (PNAAL) exhibits high binding specificity for its target sequence. The PNAAL pre-bound to the A-loop sequence prevents tRNA3(Lys) priming on the viral RNA consequently blocking in vitro initiation of reverse transcription. Further, PNAAL can efficiently disrupt the preformed [tRNA3(Lys)--viral RNA] complex thereby rendering it non-functional for reverse transcription. The endogenous reverse transcription in disrupted HIV-1 virions containing packaged tRNA3(Lys) and its replicating enzyme RT was significantly inhibited by PNAAL, thus providing direct evidence of the involvement of the A-loop region of viral RNA genome in tRNA3(Lys) priming process. These findings suggest the potential of the A-loop region as a critical target for blocking HIV-1 replication.

Inhibition of Tat-mediated transactivation of HIV-1 LTR transcription by polyamide nucleic acid targeted to TAR hairpin element.

Tat, an essential human immunodeficiency virus type 1 protein interacts with the transactivation response element (TAR) and stimulates transcription from the viral long-terminal repeat (LTR). Blockage of Tat-TAR interaction halts viral transcription and hence replication. We have found that polyamide nucleic acid (PNA), targeted to the TAR sequences of viral RNA genome is able to prevent Tat-TAR interaction by efficient sequestration of the TAR. Anti-TAR PNA competes for TAR and prevents Tat-mediated stimulation of HIV-1 LTR transcription in vitro but has no influence on the basal level of transcription in the absence of Tat. Using a reporter gene construct pHIV LTR-CAT and pCMV-Tat in cell culture, we have further shown that anti-TAR PNA is able to block Tat-mediated transactivation of HIV-1 LTR transcription in vivo as judged by the extent of LTR driven CAT gene expression in the absence and presence of anti-TAR PNA. Supplementation of 100 nM of anti-TAR PNA into the culture medium further enhances the suppression of transactivation. Nonspecific scrambled PNA had no influence on Tat-TAR interaction and LTR-driven CAT gene expression in cell culture. These results suggest that PNA targeted to the TAR sequence of the viral genome may be a potential inhibitor of HIV-1 gene expression.

Fmoc/Acyl protecting groups in the synthesis of polyamide (peptide) nucleic acid monomers

The chemical synthesis of polyamide (peptide) nucleic acid (PNA) monomers 22–25 has been accomplished using Fmoc [N-(2-aminoethyl)glycine backbone], anisoyl (adenine), 4-tert-butylbenzoyl (cytosine) and isobutyryl/diphenylcarbamoyl (guanine) protecting-group combinations, thus allowing oligomer synthesis on both peptide and oligonucleotide synthesizers. An alternative method for the preparation of (N 6-anisoyladenin-9-yl)acetic acid 7 is described using partial hydrolysis of a dianisoylated derivative. Different methods were studied for guanine alkylation including (a) Mitsunobu reaction; (b) low-temperature, sodium hydride- and (c) N,N-diisopropylethylamine-mediated alkylation reactions to give preferentially N 9-substituted derivatives. Empirical rules are proposed for differentiating N 9/N 7-substituted guanines based on their 13C NMR chemical-shift differences.

Polyamide nucleic acid-DNA chimera lacking the phosphate backbone are novel primers for polymerase reaction catalyzed by DNA polymerases.

A peptide nucleic acid (PNA) oligomer, an analogue of DNA, was examined for its ability to function as a primer or a template to support DNA synthesis catalyzed by DNA polymerases. The analogue, (PNA)19-TpG-OH, comprised of 19 bases in the form of PNA followed by a dinucleotide (TpG-OH) with a single phosphate and a free 3'OH terminus, was recognized as a bona fide primer by 2 reverse transcriptases and also by the Klenow fragment of E. coli DNA polymerase I. The 21-mer PNA chimera is extended on both RNA and DNA templates by all three polymerases. The specificity of binding of the PNA chimeric primer/DNA template at the template-primer binding site of the enzyme was shown by its photo-cross-linking ability to the enzyme which could be effectively competed out by another TP but not by template or primer alone. Furthermore, the chimeric TP-enzyme covalent complex was found to be catalytically active as judged by its ability to incorporate one nucleotide onto the 3'OH terminus of the immobilized primer. PNA sequences were also recognized as template when annealed with a DNA primer. These observations are in variance with the conventional suggestion that the phosphate backbone in the duplex region is essential for recognition and binding by DNA polymerases. The efficient extension of (PNA)19-TpG-OH suggests that the diameter of the duplex region of template primer rather than the phosphate backbone may be sufficient for recognition by DNA polymerases.

Polyamide nucleic acid targeted to the primer binding site of the HIV-1 RNA genome blocks in vitro HIV-1 reverse transcription.

We report here that polyamide nucleic acid (PNA) as well as a polyamide nucleic acid-DNA chimera complementary to the primer binding site of the HIV-1 genome can completely block priming by tRNA3Lys and consequently the in vitro initiation of reverse transcription by HIV-1 RT. Conventional heating and cooling is not required for annealing PNA analogs to the complementary nucleotide sequence as effective blockage of reverse transcription results from their invasion in the duplex region of preprimed U5-PBS HIV-1 RNA template-primer and was seen even at ambient temperature. Further, the extension of the initiated nascent (-) strand DNA can also be blocked by inclusion of another PNA, targeted to upstream sequences in the U5 region of the viral RNA. Interestingly, a PNA chimera having only two DNA nucleotides annealed with the U5-PBS RNA is recognized as a bonafide primer by HIV-1 RT, as the 3'OH end of the chimeric molecule is extended by the enzyme in the presence of dNTPs. A significant observation was that RNA/PNA or RNA/(PNA-DNA) hybrids were entirely resistant to the RNase H activity of HIV-1 RT. Furthermore, PNA invasion into the RNA/DNA hybrid completely prevented the cleavage of the RNA strand, suggesting that the RNase H activity of HIV-1 RT which was required in reverse transcription may also be inhibited by the PNA oligomer. These observations suggest that oligomeric PNAs targeted to various critical regions of the viral genome are likely to have strong therapeutic potential for interrupting multiple steps involved in the replication of HIV-1 and warrant serious investigation especially in the area of an effective delivery system.

Liquid Phase Synthesis of Peptide Nucleic Acid (or Polyamide Nucleic Acid) Dimers

The liquid phase synthesis of “polyamide nucleic acid” (PNA) dimers containing the purine nucleic acid bases adenine and guanine has been achieved in good yields. This strategy was elaborated in order to circumvent difficult direct coupling of protected PNA monomers. This method can be applied to the liquid phase synthesis of short protected polyPNAs fragments, which can then selectively be deprotected.

Hybridization Studies with Chiral Peptide Nucleic Acids

A novel class of chiral peptide nucleic acids has been synthesized in which the sugar-phosphate backbone of DNA has been replaced with the glycyl-proline backbone of both theL- and theD-configurations, nucleobases being attached through the 4-position of proline withcis- andtrans-stereochemistry. TheT10homopolymers withcis-stereochemistry in theL- andD-series bind strongly to poly(dA) withTmvalues of 69 and 70°C, respectively. They bind more strongly to poly(rA) withTmvalues of 73 and 72°C, respectively, and with apparent 1:1 stoichiometry. Using a mixed sequence decamer it was found that the thermal stability of the chiral peptide nucleic acid/oligonucleotide complex was comparable to that formed by Nielsen's polyamide nucleic acid.

Vector-mediated delivery of a polyamide ("peptide") nucleic acid analogue through the blood-brain barrier in vivo.

Polyamide ("peptide") nucleic acids (PNAs) are molecules with antigene and antisense effects that may prove to be effective neuropharmaceuticals if these molecules are enabled to undergo transport through the brain capillary endothelial wall, which makes up the blood-brain barrier in vivo. The model PNA used in the present studies is an 18-mer that is antisense to the rev gene of human immunodeficiency virus type 1 and is biotinylated at the amino terminus and iodinated at a tyrosine residue near the carboxyl terminus. The biotinylated PNA was linked to a conjugate of streptavidin (SA) and the OX26 murine monoclonal antibody to the rat transferrin receptor. The blood-brain barrier is endowed with high transferrin receptor concentrations, enabling the OX26-SA conjugate to deliver the biotinylated PNA to the brain. Although the brain uptake of the free PNA was negligible following intravenous administration, the brain uptake of the PNA was increased at least 28-fold when the PNA was bound to the OX26-SA vector. The brain uptake of the PNA bound to the OX26-SA vector was 0.1% of the injected dose per gram of brain at 60 min after an intravenous injection, approximating the brain uptake of intravenously injected morphine. The PNA bound to the OX26-SA vector retained the ability to bind to synthetic rev mRNA as shown by RNase protection assays. In summary, the present studies show that while the transport of PNAs across the blood-brain barrier is negligible, delivery of these potential neuropharmaceutical drugs to the brain may be achieved by coupling them to vector-mediated peptide-drug delivery systems.

Kinetics and mechanism of polyamide ("peptide") nucleic acid binding to duplex DNA.

To elucidate the mechanism of recognition of double-stranded DNA (dsDNA) by homopyrimidine polyamide ("peptide") nucleic acid (PNA) leading to the strand-displacement, the kinetics of the sequence-specific PNA/DNA binding have been studied. The binding was monitored with time by the gel retardation and nuclease S1 cleavage assays. The experimental kinetic curves obey pseudo-first-order kinetics and the dependence of the pseudo-first-order rate constant, kps, on PNA concentration, P, obeys a power law kps approximately P gamma with 2 < gamma < 3. The kps values for binding of decamer PNA to dsDNA target sites with one mismatch are hundreds of times slower than for the correct site. A detailed kinetic scheme for PNA/DNA binding is proposed that includes two major steps of the reaction of strand invasion: (i) a transient partial opening of the PNA binding site on dsDNA and incorporation of one PNA molecule with the formation of an intermediate PNA/DNA duplex and (ii) formation of a very stable PNA2/DNA triplex. A simple theoretical treatment of the proposed kinetic scheme is performed. The interpretation of our experimental data in the framework of the proposed kinetic scheme leads to the following conclusions. The sequence specificity of the recognition is essentially provided at the "search" step of the process, which consists in the highly reversible transient formation of duplex between one PNA molecule and the complementary strand of duplex DNA while the other DNA strand is displaced. This search step is followed by virtually irreversible "locking" step via PNA2/DNA triplex formation. The proposed mechanism explains how the binding of homopyrimidine PNA to dsDNA meets two apparently mutually contradictory features: high sequence specificity of binding and remarkable stability of both correct and mismatched PNA/DNA complexes.

Molecular mechanics calculations of the structures of polyamide nucleic acid DNA duplexes and triple helical hybrids.

Polyamide nucleic acids (PNAs) have emerged as useful agents for recognition of single- and double-stranded nucleic acids. Interresidue hydrogen bonds between the amide carbonyl nearest the nucleobase and chain NH moieties provide inherent stability to the helical conformation of PNA 1. Moving the amide carbonyl away from the nucleobase to the backbone, and replacing it with a methylene group, results in 2 lacking the stabilizing hydrogen bond. Oligomers of 2 do not interact with DNA. Modeling suggests that 2 displays a more extended conformation than 1, and nucleobase orientation is disrupted in 2 in the absence of a complementary DNA strand. This is in contrast to 1, which retains a centrosymmetric arrangement of nucleobases. Structures for 1-T10.DNA and (1-T10)2.DNA species spanned by a pyrimidine strand (D-loop) were constructed. In the triple helical (1-T10)2.DNA structure, the two PNA strands form the complementary Watson-Crick paired strand and the Hoogsteen base-paired strand in the major groove of the 1.DNA duplex. The PNA strands are proposed to bind antiparallel to one another in (1-T10)2.DNA structure. The factors suggested to account for the stability of this 2:1 complex are (i) a hydrophobic attraction between two PNA backbones and (ii) a favorable electrostatic effect resulting from replacement of a phosphodiester backbone by a neutral peptide backbone.

DNA unwinding upon strand-displacement binding of a thymine-substituted polyamide to double-stranded DNA.

It was recently found that polyamide nucleic acid (PNA) analogues consisting of thymines attached to an aminoethylglycine backbone bind strongly and sequence-selectively to adenine sequences of oligonucleotides and double-stranded DNA [Nielsen, P. E., Egholm, M., Berg, R. H. & Buchardt, O. (1991) Science 254, 1497-1500]. It was concluded that the binding to double-stranded DNA was accomplished via strand displacement, in which the PNA bound to the Watson-Crick complementary adenine-containing strand, whereas the thymine-containing strand was extruded in a virtually single-stranded conformation. This model may provide a general way in which to obtain sequence-specific recognition of any sequence in double-stranded DNA by Watson-Crick hydrogen-bonding base-pair recognition, and it is thus paramount to rigorously establish this binding mode for synthetic DNA-binding ligands. We now report such results from electron microscopy. Furthermore, we show that binding of PNA to closed circular DNA results in unwinding of the double helix corresponding to approximately one turn of the double helix per 10 base pairs. The DNA.PNA complex, which is formed at low salt concentration (only a small portion of DNA molecules show complex formation at NaCl concentration higher than 40 mM), is exceptionally kinetically stable and cannot be dissociated by increasing salt concentration up to 500 mM.

Cell Delivery and Mechanisms of Action of Antisense Oligonucleotides

Publisher Summary This chapter discusses the synthetic nucleic acids interfering with gene expression (SNAIGE), which are being developed as specific regulators of gene expression both for fundamental studies and/or for potential (medical) applications. Although straightforward in principle, the approach still faces many unknowns. Cell-free studies with model systems have helped one to learn much about the various modes through which oligonucleotides bind to their nucleic acids or protein targets. Increasing numbers of modifications allowing improved resistance toward nucleases or more efficient interaction with various targets have been devised and more will be forthcoming. A most exciting prospect has been enabled by synthetic material; the normal backbone of DNA has been replaced by a polyamide linkage, giving rise to “polyamide nucleic acid” that retains specificity in base recognition. Although a large number of genes have been successfully down-regulated in various biological systems. The tools of cell biology will help in solving pending questions, such as intracellular routing of SNAIGE, involvement of RNase H in their action, and accessibility of various sites in mRNAs, pre-mRNAs, or DNAs in their cellular environment. This helps to devise new tools on a more rational basis and to choose the best strategy in each case.

Sequence-Selective Recognition of DNA by Strand Displacement with a Thymine-Substituted Polyamide

A polyamide nucleic acid (PNA) was designed by detaching the deoxyribose phosphate backbone of DNA in a computer model and replacing it with an achiral polyamide backbone. On the basis of this model, oligomers consisting of thymine-linked aminoethylglycyl units were prepared. These oligomers recognize their complementary target in double-stranded DNA by strand displacement. The displacement is made possible by the extraordinarily high stability of the PNA-DNA hybrids. The results show that the backbone of DNA can be replaced by a polyamide, with the resulting oligomer retaining base-specific hybridization.