Development of the Right- and Left-Handed Gamma Peptide Nucleic Acid Building Blocks for On-Resin Chemical Functionalization.

Gamma peptide nucleic acids (PNAs) are a promising class of nucleic acid mimics that adopt either a right- or left-handed helical motif as individual strands and hybridize to DNA or RNA with high affinity and sequence specificity, or not at all, depending on the helical sense. They are attractive as antisense and antigene reagents, as well as building blocks for molecular self-assembly; however, they have not been widely adopted due to their relatively poor biophysical attributes and the challenge in chemical modifications. Here, we report the development of a set of universal monomers, four each for both the right- and left-handed conformers, that permit rapid and selective on-resin chemical functionalization and diversification. The system is modular, permitting incorporation of different chemical groups in the backbone without causing adverse effects on hybridization. The approach overcomes the need to prepare a new set of monomers each time a different chemical group is introduced in the backbone. The newly added synthetic flexibility, along with superior hybridization property, recognition orthogonality, and helical sense translational capability, significantly expands the scope of gamma PNA in biology, biotechnology, and molecular engineering.

Rapid self-assembly of γPNA nanofibers at constant temperature.

Peptide nucleic acids (PNAs) have primarily been used to achieve therapeutic gene modulation through antisense strategies since their design in the 1990s. However, the application of PNAs as a functional nanomaterial has been more recent. We recently reported that γ-modified peptide nucleic acids (γPNAs) could be used to enable formation of complex, self-assembling nanofibers in select polar aprotic organic solvent mixtures. Here we demonstrate that distinct γPNA strands, each with a high density of γ-modifications can form complex nanostructures at constant temperatures within 30 minutes. Additionally, we demonstrate DNA-assisted isothermal growth of γPNA nanofibers, thereby overcoming a key hurdle for future scale-up of applications related to nanofiber growth and micropatterning.

The NIH Somatic Cell Genome Editing program.

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.

Bifunctional small molecule-oligonucleotide hybrid as microRNA inhibitor.

miRNAs are key regulators of various biological processes. Dysregulation of miRNA is linked to many diseases. Development of miRNA inhibitor has implication in disease therapy and study of miRNA function. The biogenesis pathway of miRNA involves the processing of pre-miRNA into mature miRNA by Dicer enzyme. We previously reported a proximity enabled approach that employs bifunctional small molecules to regulate miRNA maturation through inhibiting the enzymatic activity of Dicer. By conjugating to an RNA targeting unit, an RNase inhibitor could be delivered to the cleavage site of specific pre-miRNA to deactivate the complexed Dicer enzyme. Herein, we expanded this bifunctional strategy by showing that antisense oligonucleotides (ASOs), including morpholinos and γPNAs, could be readily used as the RNA recognition unit to generate bifunctional small molecule-oligonucleotide hybrids as miRNA inhibitors. A systematic comparison revealed that the potency of these hybrids is mainly determined by the RNA binding of the targeting ASO molecules. Since the lengths of the ASO molecules used in this approach were much shorter than commonly used anti-miRNA ASOs, this may provide benefits to the specificity and cellular delivery of these hybrids. We expect that this approach could be complementary to traditional ASO and small molecule based miRNA inhibition and contribute to the study of miRNA.

A Robust Method for Preparing Optically Pure MiniPEG-Containing Gamma PNA Monomers.

We report the syntheses of chemical building blocks of a particular class of chiral PNAs, called miniPEG-containing (R)-gamma PNAs (or (R)-MPγPNAs). The strategy involves the application of 9-(4-bromophenyl)-9-fluorenyl as a temporary, safety-catch protecting group for the suppression of racemization in the alkylation and reductive amination steps. The methodology is general and robust, ideally suited for large-scale monomer productions with most synthetic steps providing excellent chemical yields without the need for purification other than a simple workup and precipitation.

Synthesis of ( R)- and ( S)-Fmoc-Protected Diethylene Glycol Gamma PNA Monomers with High Optical Purity.

A robust synthetic route has been developed for preparing optically pure, Fmoc-protected diethylene glycol-containing ( R)- and ( S)-γPNA monomers. The strategy involves the application of 9-(4-bromophenyl)-9-fluorenyl as a temporary, safety-catch protecting group for the suppression of epimerization in the O-alkylation and reductive amination steps. The optical purities of the final monomers were determined to be greater than 99.5% ee, as assessed by 19F-NMR and HPLC. The new synthetic methodology is well-suited for large-scale monomer production, with most synthetic steps providing excellent chemical yields without the need for chromatographic purification other than a simple workup and precipitation.

Orthogonal γPNA Dimerization Domains Empower DNA Binders with Cooperativity and Versatility Mimicking that of Transcription Factor Pairs.

Synthetic molecules capable of DNA binding and mimicking cooperation of transcription factor (TF) pairs have long been considered a promising tool for manipulating gene expression. Our previously reported Pip-HoGu system, a programmable DNA binder pyrrole-imidazole polyamides (PIPs) conjugated to host-guest moiety, defined a general framework for mimicking cooperative TF pair-DNA interactions. Here, we supplanted the cooperation modules with left-handed (LH) γPNA modules: i.e., PIPs conjugated with nucleic acid-based cooperation system (Pip-NaCo). LH γPNA was chosen because of its bioorthogonality, sequence-specific interaction, and high binding affinity toward the partner strand. From the results of the Pip-NaCo system, cooperativity is highly comparable to the natural TF pair-DNA system, with a minimum energetics of cooperation of -3.27 kcal mol-1 . Moreover, through changing the linker conjugation site, binding mode, and the length of γPNAs sequence, the cooperative energetics of Pip-NaCo can be tuned independently and rationally. The current Pip-NaCo platform might also have the potential for precise manipulation of biological processes through the construction of triple to multiple heterobinding systems.

In utero nanoparticle delivery for site-specific genome editing.

Genetic diseases can be diagnosed early during pregnancy, but many monogenic disorders continue to cause considerable neonatal and pediatric morbidity and mortality. Early intervention through intrauterine gene editing, however, could correct the genetic defect, potentially allowing for normal organ development, functional disease improvement, or cure. Here we demonstrate safe intravenous and intra-amniotic administration of polymeric nanoparticles to fetal mouse tissues at selected gestational ages with no effect on survival or postnatal growth. In utero introduction of nanoparticles containing peptide nucleic acids (PNAs) and donor DNAs corrects a disease-causing mutation in the ß-globin gene in a mouse model of human ß-thalassemia, yielding sustained postnatal elevation of blood hemoglobin levels into the normal range, reduced reticulocyte counts, reversal of splenomegaly, and improved survival, with no detected off-target mutations in partially homologous loci. This work may provide the basis for a safe and versatile method of fetal gene editing for human monogenic disorders.

Interaction of α-Thymidine Inhibitors with Thymidylate Kinase from Plasmodium falciparum.

Plasmodium falciparum thymidylate kinase (PfTMK) is a critical enzyme in the de novo biosynthesis pathway of pyrimidine nucleotides. N-(5'-Deoxy-α-thymidin-5'-yl)- N'-[4-(2-chlorobenzyloxy)phenyl]urea was developed as an inhibitor of PfTMK and has been reported as an effective inhibitor of P. falciparum growth with an EC50 of 28 nM [Cui, H., et al. (2012) J. Med. Chem. 55, 10948-10957]. Using this compound as a scaffold, a number of derivatives were developed and, along with the original compound, were characterized in terms of their enzyme inhibition ( Ki) and binding affinity ( KD). Furthermore, the binding site of the synthesized compounds was investigated by a combination of mutagenesis and docking simulations. Although the reported compound is indicated to be highly effective in its inhibition of parasite growth, we observed significantly lower binding affinity and weaker inhibition of PfTMK than expected from the reported EC50. This suggests that significant structural optimization will be required for the use of this scaffold as an effective PfTMK inhibitor and that the inhibition of parasite growth is due to an off-target effect.

Design of Bivalent Nucleic Acid Ligands for Recognition of RNA-Repeated Expansion Associated with Huntington's Disease.

We report the development of a new class of nucleic acid ligands that is comprised of Janus bases and the MPγPNA backbone and is capable of binding rCAG repeats in a sequence-specific and selective manner via, inference, bivalent H-bonding interactions. Individually, the interactions between ligands and RNA are weak and transient. However, upon the installation of a C-terminal thioester and an N-terminal cystine and the reduction of disulfide bond, they undergo template-directed native chemical ligation to form concatenated oligomeric products that bind tightly to the RNA template. In the absence of an RNA target, they self-deactivate by undergoing an intramolecular reaction to form cyclic products, rendering them inactive for further binding. The work has implications for the design of ultrashort nucleic acid ligands for targeting rCAG-repeat expansion associated with Huntington's disease and a number of other related neuromuscular and neurodegenerative disorders.

Design of a "Mini" Nucleic Acid Probe for Cooperative Binding of an RNA-Repeated Transcript Associated with Myotonic Dystrophy Type 1.

Toxic RNAs containing expanded trinucleotide repeats are the cause of many neuromuscular disorders, one being myotonic dystrophy type 1 (DM1). DM1 is triggered by CTG-repeat expansion in the 3'-untranslated region of the DMPK gene, resulting in a toxic gain of RNA function through sequestration of MBNL1 protein, among others. Herein, we report the development of a relatively short miniPEG-γ peptide nucleic acid probe, two triplet repeats in length, containing terminal pyrene moieties, that is capable of binding rCUG repeats in a sequence-specific and selective manner. The newly designed probe can discriminate the pathogenic rCUGexp from the wild-type transcript and disrupt the rCUGexp-MBNL1 complex. The work provides a proof of concept for the development of relatively short nucleic acid probes for targeting RNA-repeat expansions associated with DM1 and other related neuromuscular disorders.

RNA-Templated Concatenation of Triplet Nucleic-Acid Probe.

Template-directed synthesis offers several distinct benefits over conventional laboratory creation, including unsurpassed reaction rate and selectivity. Although it is central to many biological processes, such an approach has rarely been applied to the in situ synthesis and recognition of biomedically relevant target. Towards this goal, we report the development of a three-codon nucleic-acid probe containing a C-terminal thioester group and an N-terminal cysteine that is capable of undergoing template-directed oligomerization in the presence of an RNA target and self-deactivation in its absence. The work has implications for the development of millamolecular nucleic-acid probes for targeting RNA-repeated expansions associated with myotonic dystrophy type 1 and other related neuromuscular and neurodegenerative disorders.

Shape selective bifacial recognition of double helical DNA.

An impressive array of antigene approaches has been developed for recognition of double helical DNA over the past three decades; however, few have exploited the 'Watson-Crick' base-pairing rules for establishing sequence-specific recognition. One approach employs peptide nucleic acid as a molecular reagent and strand invasion as a binding mode. However, even with integration of the latest conformationally-preorganized backbone design, such an approach is generally confined to sub-physiological conditions due to the lack of binding energy. Here we report the use of a class of shape-selective, bifacial nucleic acid recognition elements, namely Janus bases, for targeting double helical DNA or RNA. Binding occurs in a highly sequence-specific manner under physiologically relevant conditions. The work may provide a foundation for the design of oligonucleotides for targeting the secondary and tertiary structures of nucleic acid biopolymers.

Anti-tumor Activity of miniPEG-γ-Modified PNAs to Inhibit MicroRNA-210 for Cancer Therapy.

MicroRNAs (miRs) are frequently overexpressed in human cancers. In particular, miR-210 is induced in hypoxic cells and acts to orchestrate the adaptation of tumor cells to hypoxia. Silencing oncogenic miRs such as miR-210 may therefore offer a promising approach to anticancer therapy. We have developed a miR-210 inhibition strategy based on a new class of conformationally preorganized antisense γ peptide nucleic acids (γPNAs) that possess vastly superior RNA-binding affinity, improved solubility, and favorable biocompatibility. For cellular delivery, we encapsulated the γPNAs in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). Our results show that γPNAs targeting miR-210 cause significant delay in growth of a human tumor xenograft in mice compared to conventional PNAs. Further, histopathological analyses show considerable necrosis, fibrosis, and reduced cell proliferation in γPNA-treated tumors compared to controls. Overall, our work provides a chemical framework for a novel anti-miR therapeutic approach using γPNAs that should facilitate rational design of agents to potently inhibit oncogenic microRNAs.

In vivo correction of anaemia in ß-thalassemic mice by γPNA-mediated gene editing with nanoparticle delivery.

The blood disorder, ß-thalassaemia, is considered an attractive target for gene correction. Site-specific triplex formation has been shown to induce DNA repair and thereby catalyse genome editing. Here we report that triplex-forming peptide nucleic acids (PNAs) substituted at the γ position plus stimulation of the stem cell factor (SCF)/c-Kit pathway yielded high levels of gene editing in haematopoietic stem cells (HSCs) in a mouse model of human ß-thalassaemia. Injection of thalassemic mice with SCF plus nanoparticles containing γPNAs and donor DNAs ameliorated the disease phenotype, with sustained elevation of blood haemoglobin levels into the normal range, reduced reticulocytosis, reversal of splenomegaly and up to 7% ß-globin gene correction in HSCs, with extremely low off-target effects. The combination of nanoparticle delivery, next generation γPNAs and SCF treatment may offer a minimally invasive treatment for genetic disorders of the blood that can be achieved safely and simply by intravenous administration.

Gamma Peptide Nucleic Acids: As Orthogonal Nucleic Acid Recognition Codes for Organizing Molecular Self-Assembly.

Nucleic acids are an attractive platform for organizing molecular self-assembly because of their specific nucleobase interactions and defined length scale. Routinely employed in the organization and assembly of materials in vitro, however, they have rarely been exploited in vivo, due to the concerns for enzymatic degradation and cross-hybridization with the host's genetic materials. Herein we report the development of a tight-binding, orthogonal, synthetically versatile, and informationally interfaced nucleic acid platform for programming molecular interactions, with implications for in vivo molecular assembly and computing. The system consists of three molecular entities: the right-handed and left-handed conformers and a nonhelical domain. The first two are orthogonal to each other in recognition, while the third is capable of binding to both, providing a means for interfacing the two conformers as well as the natural nucleic acid biopolymers (i.e., DNA and RNA). The three molecular entities are prepared from the same monomeric chemical scaffold, with the exception of the stereochemistry or lack thereof at the γ-backbone that determines if the corresponding oligo adopts a right-handed or left-handed helix, or a nonhelical motif. These conformers hybridize to each other with exquisite affinity, sequence selectivity, and level of orthogonality. Recognition modules as short as five nucleotides in length are capable of organizing molecular assembly.

Synthesis of optically pure γPNA monomers: a comparative study.

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.

Single-stranded γPNAs for in vivo site-specific genome editing via Watson-Crick recognition.

Triplex-forming peptide nucleic acids (PNAs) facilitate gene editing by stimulating recombination of donor DNAs within genomic DNA via site-specific formation of altered helical structures that further stimulate DNA repair. However, PNAs designed for triplex formation are sequence restricted to homopurine sites. Herein we describe a novel strategy where next generation single-stranded gamma PNAs (γPNAs) containing miniPEG substitutions at the gamma position can target genomic DNA in mouse bone marrow at mixed-sequence sites to induce targeted gene editing. In addition to enhanced binding, γPNAs confer increased solubility and improved formulation into poly(lactic-co-glycolic acid) (PLGA) nanoparticles for efficient intracellular delivery. Single-stranded γPNAs induce targeted gene editing at frequencies of 0.8% in mouse bone marrow cells treated ex vivo and 0.1% in vivo via IV injection, without detectable toxicity. These results suggest that γPNAs may provide a new tool for induced gene editing based on Watson-Crick recognition without sequence restriction.

Cooperative hybridization of γPNA miniprobes to a repeating sequence motif and application to telomere analysis.

GammaPNA oligomers having one or two repeats of the sequence AATCCC were designed to hybridize to DNA having one or more repeats of the complementary TTAGGG sequence found in the human telomere. UV melting curves and surface plasmon resonance experiments demonstrate high affinity and cooperativity for hybridization of these miniprobes to DNA having multiple complementary repeats. Fluorescence spectroscopy for Cy3-labeled miniprobes demonstrate increases in fluorescence intensity for assembling multiple short probes on a DNA target compared with fewer longer probes. The fluorescent γPNA miniprobes were then used to stain telomeres in metaphase chromosomes derived from U2OS cells possessing heterogeneous long telomeres and Jurkat cells harboring homogenous short telomeres. The miniprobes yielded comparable fluorescence intensity to a commercially available PNA 18mer probe in U2OS cells, but significantly brighter fluorescence was observed for telomeres in Jurkat cells. These results suggest that γPNA miniprobes can be effective telomere-staining reagents with applications toward analysis of critically short telomeres, which have been implicated in a range of human diseases.

MiniPEG-γPNA.

Peptide nucleic acids (PNAs) are attractive, as compared to other classes of oligonucleotides that have been developed to date, in that they are relatively easy to synthesize and modify, hybridize to DNA and RNA with high affinity and sequence selectivity, and are resistant to enzymatic degradation by proteases and nucleases; however, the downside is that they are only moderately soluble in aqueous solution. Herein we describe the protocols for synthesizing the second-generation γPNAs, both the monomers and oligomers, containing MiniPEG side chain with considerable improvements in water solubility, biocompatibility, and hybridization properties.