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PDF file - 2.2MB, Modified STAT3 decoys demonstrate enhanced resistance to thermal denaturation

PDF file - 1.2MB, Modified STAT3 decoys bind to pSTAT3 protein with similar affinity as the parental STAT3 decoy

PDF file - 107K, Systemic delivery of cyclic STAT3 decoy does not affect total or pSTAT3 expression levels

PDF file - 2.5MB, The chemistry used to generate the modified decoys

PDF file - 70K, Lack of anti-tumor effects of systemically administered parental STAT3 decoy

PDF file - 2MB, Modified STAT3 decoys abrogate STAT3 target gene expression in vitro

PDF file - 165K, EC50 values of STAT3 decoy in cancer cell lines

PDF file - 70K

We report a systematic study examining two synthetic routes, reductive amination and Mitsunobu coupling, for preparation of chiral γ-peptide nucleic acid (γPNA) monomers and oligomers. We found that the reductive amination route is prone to epimerization, even under mild experimental conditions. The extent of epimerization could be minimized by utilizing a bulky protecting group such as PhFl; however, it is difficult to remove in the subsequent oligomer synthesis stage. On the other hand, we found that the Mitsunobu route produced optically superior products using standard carbamate protecting groups.

Peptide nucleic acids have emerged over the past two decades as a promising class of nucleic acid mimics because of their strong binding affinity and sequence selectivity toward DNA and RNA, and resistance to enzymatic degradation by proteases and nucleases. While they have been shown to be effective in regulation of gene expression in vitro, and to a small extent in vivo, their full potential for molecular therapy has not yet been fully realized due to poor cellular uptake. Herein, we report the development of cell-permeable, guanidine-based peptide nucleic acids targeting the epidermal growth factor receptor (EGFR) in preclinical models as therapeutic modality for head and neck squamous cell carcinoma (HNSCC) and nonsmall cell lung cancer (NSCLC). A GPNA oligomer, 16 nucleotides in length, designed to bind to EGFR gene transcript elicited potent antisense effects in HNSCC and NSCLC cells in preclinical models. When administered intraperitoneally in mice, EGFRAS-GPNA was taken-up by several tissues including the xenograft tumor. Systemic administration of EGFRAS-GPNA induced antitumor effects in HNSCC xenografts, with similar efficacies as the FDA-approved EGFR inhibitors: cetuximab and erlotinib. In addition to targeting wild-type EGFR, EGFRAS-GPNA is effective against the constitutively active EGFR vIII mutant implicated in cetuximab resistance. Our data reveals that GPNA is just as effective as a molecular platform for treating cetuximab resistant cells, demonstrating its utility in the treatment of cancer.

Chiral γPNAs containing miniPEG side-chains can invade any sequence of double helical B-form DNA, with the recognition occurring through direct Watson-Crick base-pairing.

Developed in the early 1990s, peptide nucleic acid (PNA) has emerged as a promising class of nucleic acid mimic because of its strong binding affinity and sequence selectivity toward DNA and RNA and resistance to enzymatic degradation by proteases and nucleases; however, the main drawbacks, as compared to other classes of oligonucleotides, are water solubility and biocompatibility. Herein we show that installation of a relatively small, hydrophilic (R)-diethylene glycol ("miniPEG", R-MP) unit at the γ-backbone transforms a randomly folded PNA into a right-handed helix. Synthesis of optically pure (R-MP)γPNA monomers is described, which can be accomplished in a few simple steps from a commercially available and relatively cheap Boc-l-serine. Once synthesized, (R-MP)γPNA oligomers are preorganized into a right-handed helix, hybridize to DNA and RNA with greater affinity and sequence selectivity, and are more water soluble and less aggregating than the parental PNA oligomers. The results presented herein have important implications for the future design and application of PNA in biology, biotechnology, and medicine, as well as in other disciplines, including drug discovery and molecular engineering.

Peptide nucleic acids (PNAs) make up the only class of nucleic acid mimics developed to date that has been shown to be capable of invading double-helical B-form DNA. Recently, we showed that sequence limitation associated with PNA recognition can be relaxed by utilizing conformationally preorganized γ-peptide nucleic acids (γPNAs). However, like all the previous studies, with the exception of triplex binding, DNA strand invasion was performed at relatively low salt concentrations. When physiological ionic strengths were used, little to no binding was observed. On the basis of this finding, it was not clear whether the lack of binding is due to the lack of base pair opening or the lack of binding free energy, either of which would result in no productive binding. In this work, we show that it is the latter. Under simulated physiological conditions, the DNA double helix is sufficiently dynamic to permit strand invasion by the designer oligonucleotide molecules provided that the required binding free energy can be met. This finding has important implications for the design oligonucleotides for recognition of B-DNA via direct Watson-Crick base pairing.

We have determined the structure of a PNA-DNA duplex to 1.7 A resolution by multiple-wavelength anomalous diffraction phasing method on a zinc derivative. This structure represents the first high-resolution 3D view of a hybrid duplex containing a contiguous chiral PNA strand with complete gamma-backbone modification ("gammaPNA"). Unlike the achiral counterpart, which adopts a random-fold, this particular gammaPNA is already preorganized into a right-handed helix as a single strand. The new structure illustrates the unique characteristics of this modified PNA, possessing conformational flexibility while maintaining sufficient structural integrity to ultimately adopt the preferred P-helical conformation upon hybridization with DNA. The unusual structural adaptability found in the gammaPNA strand is crucial for enabling the accommodation of backbone modifications while constraining conformational states. In conjunction with NMR analysis characterizing the structures and substructures of the individual building blocks, these results provide unprecedented insights into how this new class of chiral gammaPNA is preorganized and stabilized, before and after hybridization with a cDNA strand. Such knowledge is crucial for the future design and development of PNA for applications in biology, biotechnology, and medicine.

Peptide nucleic acid (PNA) is a synthetic mimic of DNA and RNA that can recognize double-stranded B-DNA through direct Watson-Crick base-pairing. Although promising, PNA recognition is presently limited to mostly purine- and pyrimidine-rich targets, because mixed-sequence PNA, in general, does not have sufficient binding free energy to invade B-DNA. In this Article, we show that conformationally preorganized gamma-peptide nucleic acid (gamma-PNA) containing an acridine moiety covalently linked at the C-terminus can invade mixed-sequence B-DNA in a sequence-specific manner. Recognition occurs through direct Watson-Crick base-pairing. This finding is significant because it demonstrates that the same principles that guide the recognition of single-stranded DNA and RNA can also be applied to double-stranded B-DNA.

Establishing a general and effective method for regulating gene expression in mammalian systems is important for many aspects of biological and biomedical research. Herein we report the antisense activities of a cell-permeable, guanidine-based peptide nucleic acid (PNA) called GPNA. We show that a GPNA oligomer designed to bind to the transcriptional start-site of human E-cadherin gene induces potent and sequence-specific antisense effects and is less toxic to the cells than the corresponding PNA-polyarginine conjugate. GPNA confers its silencing effect by blocking protein translation. The findings reported in this study provide a molecular framework for designing the next generation cell-permeable nucleic acid mimics for regulating gene expression in live cells and intact organisms.

Despite evidence implicating transcription factors, including STAT3, in oncogenesis, these proteins have been regarded as “undruggable.” We developed a decoy targeting STAT3 and conducted a phase 0 trial. Expression levels of STAT3 target genes were decreased in head and neck cancers following injection with the STAT3 decoy compared with tumors receiving saline control. Decoys have not been amenable to systemic administration due to instability. To overcome this barrier, we linked the oligonucleotide strands using hexaethylene glycol spacers. This cyclic STAT3 decoy bound with high affinity to STAT3 protein, reduced cellular viability, and suppressed STAT3 target gene expression in cancer cells. Intravenous injection of the cyclic STAT3 decoy inhibited xenograft growth and downregulated STAT3 target genes in the tumors. These results provide the first demonstration of a successful strategy to inhibit tumor STAT3 signaling via systemic administration of a selective STAT3 inhibitor, thereby paving the way for broad clinical development. Significance: This is the first study of a STAT3-selective inhibitor in humans and the first evidence that a transcription factor decoy can be modified to enable systemic delivery. These findings have therapeutic implications beyond STAT3 to other “undruggable” targets in human cancers. Cancer Discov; 2(8); 694–705. ©2012 AACR. Read the Commentary on this article by Koppikar et al., p. 670. This article is highlighted in the In This Issue feature, p. 653.

Charge transfer (CT) properties are compared between peptide nucleic acid structures with an aminoethylglycine backbone (aeg-PNA) and those with a γ-methylated backbone (γ-PNA). The common aeg-PNA is an achiral molecule with a flexible structure, whereas γ-PNA is a chiral molecule with a significantly more rigid structure than aeg-PNA. Electrochemical measurements show that the CT rate constant through an aeg-PNA bridging unit is twice the CT rate constant through a γ-PNA bridging unit. Theoretical calculations of PNA electronic properties, which are based on a molecular dynamics structural ensemble, reveal that the difference in the CT rate constant results from the difference in the extent of backbone fluctuations of aeg- and γ-PNA. In particular, fluctuations of the backbone affect the local electric field that broadens the energy levels of the PNA nucleobases. The greater flexibility of the aeg-PNA gives rise to more broadening, and a more frequent appearance of high-CT rate conformations than in γ-PNA.

A new single-molecule approach for rapid and purely electronic discrimination among similar genes is presented. Combining solid-state nanopores and γ-modified synthetic peptide nucleic acid (γPNA) probes, we accurately barcode genes by counting the number of probes attached to each gene, and measuring their relative spacing. We illustrate our method by sensing individual genes from two highly similar human immunodeficiency virus (HIV) subtypes, demonstrating feasibility of a novel, single-molecule diagnostic platform for rapid pathogen classification.

Macrocyclic peptides with multiple disulfide cross-linkages, such as those produced by plants and those found in nonhuman primates, as components of the innate immunity, hold great promise for molecular therapy because of their broad biological activities, and high chemical, thermal and enzymatic stability. However, for some, because of their intricate spatial arrangement and elaborate interstrand cross-linkages, they are difficult to prepare de novo in large quantities and high purity, due to the nonselective nature of disulfide-bond formation. We show that the disulfide bridges of RTD-1, a member of the θ-defensin subfamily, could be replaced with noncovalent Watson-Crick hydrogen-bonds without significantly affecting its biological activities. The work provides a general strategy for engineering conformationally-rigid, cyclic peptides without the need for disulfide-bond reinforcement.

Peptide nucleic acid (PNA) is a synthetic analogue of DNA, which has the same nucleobases as DNA but typically has a backbone based on aminoethyl glycine (Aeg). PNA forms duplexes by Watson Crick hybridization. The Aeg-based PNA duplexes adopt a chiral helical structure but do not have a preferred handedness because they do not contain a chiral center. An L-lysine situated at the C-end of one or both strands of a PNA duplex causes the duplex to preferably adopt a left-handed structure. We have introduced into the PNA duplexes both a C-terminal L-lysine and one or two PNA monomers that have a γ-(S)-methyl-aminoethyl glycine backbone, which is known to induce a preference for a right-handed structure. Indeed, we found that in these duplexes the γ-methyl monomer exerts the dominant chiral induction effect causing the duplexes to adopt a right-handed structure. The chiral PNA monomer had a 2,2':6',2''-terpyridine (Tpy) ligand instead of a nucleobase and PNA duplexes that contained one or two Tpys formed [Cu(Tpy)(2)](2+) complexes in the presence of Cu(2+). The CD spectroscopy studies showed that these metal-coordinated duplexes were right-handed due to the chiral induction effect exerted by the S-Tpy PNA monomer(s) except for the cases when the [Cu(Tpy)(2)](2+) complex was formed with Tpy ligands from two different PNA duplexes. In the latter case, the metal complex bridged the two PNA duplexes and the duplexes were left-handed. The results of this study show that the preferred handedness of a ligand-modified PNA can be switched as a consequence of metal coordination to the ligand. This finding could be used as a tool in the design of functional nucleic-acid based nanostructures.

Conformationally preorganized peptide nucleic acids (PNAs) have been synthesized through backbone modifications at the γ-position, where R = alanine, valine, isoleucine, and phenylalanine side chains. The effects of these side-chains on the conformations and hybridization properties of PNAs were determined using a combination of CD and UV-Vis spectroscopic techniques. Our results show that the γ-position can accommodate varying degrees of sterically hindered side-chains, reaffirming the bimodal function of PNAs as the true hybrids of “peptides” and “nucleic acids.”

This paper presents the results of an NMR spectroscopy and distance-restrained molecular dynamics (MD) study of a gamma-methylated, palindromic, 8-base pair peptide nucleic acid (gamma-PNA) duplex. The goal of this study was to examine the impact of the gamma-backbone modification on the structure of the PNA duplex. The 2D NMR information involving the backbone methyl group, especially the NOEs between the methyl protons and those of the amide and methylene groups of the backbone, led to distance restraints useful in the elucidation of the structure of the backbone of gamma-PNA. Integration of the NOE peaks resulted in 138 inter-proton distance restraints, which were used in ten independent simulated annealing followed by 2 ns restrained MD runs. These simulations led to the conclusion that the gamma-PNA duplex adopts a general P-form helical structure similar to that observed for non-modified PNA but with a smaller base pair rise, which is an A-like helical feature, and a slight helical bending towards the major groove (PDB ID ). These properties of the gamma-PNA duplex may be induced by the gamma-methyl group. A similar effect of the methyl group was revealed by a previous NMR study of single stranded gamma-PNA [A. Dragulescu-Andrasi, S. Rapireddy, B. M. Frezza, C. Gayathri, R. R. Gil and D. H. Ly, J. Am. Chem. Soc., 2006, 128, 10258-10267]. It appears that the steric constraint exerted by the gamma-methyl on the backbone orientation is relatively independent of the base pairing and stacking and thus is likely to manifest when other substituents are introduced at the gamma-position of the PNA.

γ-PNA, a new class of peptide nucleic acids, promises to overcome previous sequence limitations of double-stranded DNA (dsDNA) targeting with PNA. To check the potential of γ-PNA, we have synthesized a biotinylated, pentadecameric γ-PNA of mixed sequence carrying three guanidinium G-clamp nucleobases. We have found that strand invasion reactions of the γ-PNA oligomer to its fully complementary target within dsDNA occurs with significantly higher binding rates than to targets containing single mismatches. Association of the PNA oligomer to mismatched targets does not go to completion but instead reaches a stationary level at or below 60%, even at conditions of very low ionic strength. Initial binding rates to both matched and mismatched targets experience a steep decrease with increasing salt concentration. We demonstrate that a linear DNA target fragment with the correct target sequence can be purified from DNA mixtures containing mismatched target or unrelated genomic DNA by affinity capture with streptavidin-coated magnetic beads. Similarly, supercoiled plasmid DNA is obtained with high purity from an initial sample mixture that included a linear DNA fragment with the fully complementary sequence. Based on the results obtained in this study we believe that γ-PNA has a great potential for specific targeting of chosen duplex DNA sites in a sequence-unrestricted fashion.

In this communication, we show that peptide nucleic acids (PNAs) with lengths of 15-20 nucleotides, when preorganized into a right-handed helix, can invade mixed-sequence double-helical B-form DNA (B-DNA). Strand invasion occurs in a highly sequence-specific manner through direct Watson-Crick base pairing. Unlike the previously developed double-duplex invasion strategy, which requires simultaneous binding of two strands of pseudocomplementary PNAs to DNA, only a single strand of gammaPNA is required for invasion in this case, and no nucleobase substitution is needed.

Nature uses Watson–Crick base pairings as a means to store and transmit genetic information because of their high fidelity. These specific A–T (or A–U) and G–C nucleobase interactions, in turn, provide chemists and biologists with a general paradigm for designing molecules to bind to DNA and RNA. With knowledge of the sequence information, one can design oligonucleotides to bind to just about any part of these biopolymeric targets simply by choosing the corresponding nucleobase sequence according to these digital base-pairing rules. Although conceptually simple, such principles in general can only be applied to the recognition of single-stranded DNA or RNA, but not the double-stranded form. The reason is that in double-helical DNA (or RNA) not only are the Watson–Crick faces of the nucleobases already occupied, they are buried within the double helix. [1] Such molecular encapsulation imposes a steep energetic barrier on the designer molecules. To establish binding, not only would they need to be able to gain access to the designated nucleobase targets, which are blocked by the existing base pairs, they would also need to be able to compete with the complementary DNA strand to prevent it from re-annealing with its partner—a task that has rarely been accomplished by any class of molecules. To circumvent this challenge, most of the research effort to date has been focused on establishing principles for recognizing chemical groups in the minor and major groove instead because they are more readily accessible and energetically less demanding. [2] While impressive progress has been made on this front, especially in the development of triplex-forming oligonucleotides, [3–5] polyamides, [6–8] and zinc-finger-binding pep

Peptide nucleic acid (PNA) is a synthetic analogue of DNA and RNA, developed more than a decade ago in which the naturally occurring sugar phosphate backbone has been replaced by the N-(2-aminoethyl) glycine units. Unlike DNA or RNA in the unhybridized state (single strand) which can adopt a helical structure through base-stacking, although highly flexible, PNA does not have a well-defined conformational folding in solution. Herein, we show that a simple backbone modification at the gamma-position of the N-(2-aminoethyl) glycine unit can transform a randomly folded PNA into a helical structure. Spectroscopic studies showed that helical induction occurs in the C- to N-terminal direction and is sterically driven. This finding has important implication not only on the future design of nucleic acid mimics but also on the design of novel materials, where molecular organization and efficient electronic coupling are desired.

This study was aimed to develop a transcription factor decoy for efficient systemic administration in head and neck cancer. Signal Transducer and Activator of Transcription-3 (STAT3) is upregulated in numerous human cancers including head and neck squamous cell carcinoma (HNSCC). Small molecules that target STAT3 are limited by lack of specificity. We developed a transcription factor decoy targeting STAT3 consisting of a 15-mer double-stranded oligonucleotide with three phosphorothioate modifications (PTO) at the 3′ and 5′ end of the double-stranded structure. This decoy exhibits antitumor efficacy in preclinical cancer models and is now being tested in a phase 0 human trial with intratumoral inoculation. To enable systemic administration, the STAT3 decoy formulation has been modified to enhance stability and uptake into tumors. A series of chemically modified STAT3 decoys have been designed to increase resistance to nucleases and enhance stability when administered in vivo. These include tetra-nucleotide single stranded DNA sequences (DN4), 18-atom hexa-ethyleneglycol spacers (DS18), and locked nucleic acid (LNA-1) modifications to enhance stability in serum. The modified STAT3 decoys were tested for serum nuclease degradation, melting temperature, binding to pSTAT3 protein, uptake and biologic activity. LNA-1 showed increased half-life of up to 8 h in serum, whereas DN4 is stable up to 4 h, DS18 up to 3 h compared with the parent STAT3 decoy, which is resistant only up to 2 h. The melting temperature demonstrated increased thermal stability for all the modified decoys compared to the parent STAT3 decoy. The DN4, DS18 and LNA-1 modified decoy bound to recombinant STAT3 protein as well as the parent decoy and inhibited head and neck cancer cell proliferation in vitro at nanomolar concentrations. The modified STAT3 decoys (DN4, DS18 and LNA-1) showed increased uptake into head and neck cancer cells similar compared with the parent STAT3 decoy. These modified decoys will be tested for their effect on STAT3 target gene expression and in vivo antitumor efficacy when administered systemically with anticipation of implementing a phase I clinical trial if tumor growth inhibition is observed upon intravenous injection. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4139.