Targeting a KRAS i-motif forming sequence by unmodified and gamma-modified peptide nucleic acid oligomers.

Growing interest in i-motif DNA as a transcriptional regulatory element motivates development of synthetic molecules capable of targeting these structures. In this study, we designed unmodified peptide nucleic acid (PNA) and gamma-modified PNA (γPNA) oligomers complementary to an i-motif forming sequence derived from the promoter of the KRAS oncogene. Biophysical techniques such as circular dichroism (CD) spectroscopy, CD melting, and fluorescence spectroscopy demonstrated the successful invasion of the i-motif by PNA and γPNA. Both PNA and γPNA showed very strong binding to the target sequence with high thermal stability of the resulting heteroduplexes. Interestingly fluorescence and CD experiments indicated formation of an intermolecular i-motif structure via the overhangs of target-probe heteroduplexes formed by PNA/γPNA invasion of the intramolecular i-motif. Targeting promoter i-motif forming sequences with high-affinity oligonucleotide mimics like γPNAs may represent a new approach for inhibiting KRAS transcription, thereby representing a potentially useful anti-cancer strategy.

Rapid, multiplexed detection of the let-7 miRNA family using γPNA amphiphiles in micelle-tagging electrophoresis.

miRNA is a promising class of biomarkers whose levels can be assayed to detect various forms of cancer and other serious diseases. These short, noncoding nucleic acids are difficult to detect due to their low abundance and the marginal stability of their duplexes with DNA probes. In addition, miRNAs within the same family have high sequence homology, and often, related miRNA differ in sequence by only a single base. In this report, we demonstrate an independent detection seven members of the let-7 family of miRNA in a single run. Key to success is the use of mini-PEG-substituted PNA amphiphiles (γPNAA) and highly fluorescent DNA nanotags in micelle tagging electrophoresis (MTE). Multiplexed detection is accomplished in capillary electrophoresis (CE) using oligomeric nanotags of pre-programmed lengths where the presence of a specific miRNA links its nanotag to a micelle drag-tag, which shifts the nanotag elution time to a defined region for detection. We further demonstrate that the peak shape and elution time are unaffected by the presence of up to 10 mg/ml of serum protein in the sample, with a total runtime of less than 4 min and a LOD of 10-100 pM. We discuss efforts to substantially decrease the detection limit using nanotags that are >1000 bp in length.

Targeting a Potential G-Quadruplex Forming Sequence Found in the West Nile Virus Genome by Complementary Gamma-Peptide Nucleic Acid Oligomers.

In the United States, West Nile virus (WNV) infects approximately 2500 people per year, of which 100-200 cases are fatal. No antiviral drug or vaccine is currently available for WNV. In this study, we designed gamma-modified peptide nucleic acid (γPNA) oligomers to target a newly identified guanine-rich gene sequence in the WNV genome. The target is found in the NS5 protein-coding region and was previously predicted to fold into a G-quadruplex (GQ) structure. Biophysical techniques such as UV melting analysis, circular dichroism spectroscopy, and fluorescence spectroscopy demonstrated that the target RNA indeed folds into a moderately stable GQ structure at physiological temperature and potassium concentration. Successful invasion of the GQ by three complementary γPNAs was also characterized by the above-mentioned biophysical techniques. The γPNAs showed very strong binding to the target with low femtomolar affinity at physiological temperature. Targeting this potential guanine quadruplex forming sequence (PQS) and other related sequences with γPNA may represent a new approach for inhibiting both WNV replication and transcription, thereby representing a generally useful antiviral strategy.

Assembly and Characterization of RNA/DNA Hetero-G-Quadruplexes.

Transient association of guanine-rich RNA and DNA in the form of hetero-G-quadruplexes (RDQs) has emerged as an important mechanism for regulating genome transcription and replication but relatively little is known about the structure and biophysical properties of RDQs compared with DNA and RNA homo-G-quadruplexes. Herein, we report the assembly and characterization of three RDQs based on sequence motifs found in human telomeres and mitochondrial nucleic acids. Stable RDQs were assembled using a duplex scaffold, which prevented segregation of the DNA and RNA strands into separate homo-GQs. Each of the RDQs exhibited UV melting temperatures above 50 °C in 100 mM KCl and predominantly parallel morphologies, evidently driven by the RNA component. The fluorogenic dye thioflavin T binds to each RDQ with low micromolar KD values, similar to its binding to RNA and DNA homo-GQs. These results establish a method for assembling RDQs that should be amenable to screening compounds and libraries to identify selective RDQ-binding small molecules, oligonucleotides, and proteins.

Enhanced Hybridization Selectivity Using Structured GammaPNA Probes.

High affinity nucleic acid analogues such as gammaPNA (γPNA) are capable of invading stable secondary and tertiary structures in DNA and RNA targets but are susceptible to off-target binding to mismatch-containing sequences. We introduced a hairpin secondary structure into a γPNA oligomer to enhance hybridization selectivity compared with a hairpin-free analogue. The hairpin structure features a five base PNA mask that covers the proximal five bases of the γPNA probe, leaving an additional five γPNA bases available as a toehold for target hybridization. Surface plasmon resonance experiments demonstrated that the hairpin probe exhibited slower on-rates and faster off-rates (i.e., lower affinity) compared with the linear probe but improved single mismatch discrimination by up to a factor of five, due primarily to slower on-rates for mismatch vs. perfect match targets. The ability to discriminate against single mismatches was also determined in a cell-free mRNA translation assay using a luciferase reporter gene, where the hairpin probe was two-fold more selective than the linear probe. These results validate the hairpin design and present a generalizable approach to improving hybridization selectivity.

Targeting G-Quadruplexes with PNA Oligomers.

The growing interest in G-quadruplex (G4) structure and function is motivating intense efforts to develop G4-binding ligands. This chapter describes the design and testing of peptide nucleic acid (PNA) oligomers, which can bind to G4 DNA or RNA in two distinct ways, leading to formation of heteroduplexes or heteroquadruplexes. Guidelines for designing G4-targeting PNAs and step-by-step protocols for characterizing their binding through biophysical or biochemical methods are provided.

Spectral Properties of Fluorogenic Thiophene-derived Triarylmethane Dyes.

Spectral properties and fluorogenic behaviors of five novel thiophene variants of malachite green (MG), termed MGTs, were determined. Appreciable changes as a function of homologation and substitution pattern, including absorption band positions and intensities and fluorescence quantum yields were observed. In particular, the shorter wavelength y-band absorption was found to shift over a nearly 200 nm range based on aryl group variation, allowing fine-tuning of the excitation wavelength for these dyes. In addition, the fluorescence intensity of some MGTs increased significantly (up to 4600-fold) when the dye was bound to a cognate protein partner, which is potentially useful for cell imaging studies.

Efficient Cytoplasmic Delivery of Antisense Probes Assisted by Cyclized-Peptide-Mediated Photoinduced Endosomal Escape.

Intracellular delivery and endosomal release of antisense oligonucleotides remain a significant challenge in the development of gene-targeted therapeutics. Previously, noncovalently cyclized TAT peptide (Cyc-TAT), in which the final ring-closing step is accomplished by hybridization of two short complementary γPNA segments, has been proven more efficient than its linear analogues at entering cells. As Cyc-TAT also readily accommodates a binding site, that is, an overhanging γPNA sequence, for codelivery of functional nucleic acid probes into cells, we were able to demonstrate that the overhang-Cyc-TAT penetrated into A549 cells when carrying an anti-telomerase γPNA that specifically reduced telomerase activity by over 97 %. Herein, we report that the cyclized TAT(FAM) can escape endosomes much more efficiently than the linear TAT(FAM) after LED illumination (490 nm). Based on this observation, the endosomal release of overhang-Cyc-TAT(FAM)/anti-telomerase γPNA complex can be greatly enhanced by photoactivation, thus shortening cell treatment time from 60 to 3 h, while keeping the same high efficiency in inhibiting telomerase activity inside A549 cells.

Structural basis for activation of fluorogenic dyes by an RNA aptamer lacking a G-quadruplex motif.

The DIR2s RNA aptamer, a second-generation, in-vitro selected binder to dimethylindole red (DIR), activates the fluorescence of cyanine dyes, DIR and oxazole thiazole blue (OTB), allowing detection of two well-resolved emission colors. Using Fab BL3-6 and its cognate hairpin as a crystallization module, we solved the crystal structures of both the apo and OTB-SO3 bound forms of DIR2s at 2.0 Å and 1.8 Å resolution, respectively. DIR2s adopts a compact, tuning fork-like architecture comprised of a helix and two short stem-loops oriented in parallel to create the ligand binding site through tertiary interactions. The OTB-SO3 fluorophore binds in a planar conformation to a claw-like structure formed by a purine base-triple, which provides a stacking platform for OTB-SO3, and an unpaired nucleotide, which partially caps the binding site from the top. The absence of a G-quartet or base tetrad makes the DIR2s aptamer unique among fluorogenic RNAs with known 3D structure.

Closing the Loop: Constraining TAT Peptide by γPNA Hairpin for Enhanced Cellular Delivery of Biomolecules.

Based on the exceptionally high stability of γPNA duplexes, we designed a peptide/γPNA chimera in which a cell-penetrating TAT peptide is flanked by two short complementary γPNA segments. Intramolecular hybridization of the γPNA segments results in a stable hairpin conformation in which the TAT peptide is constrained to form the loop. The TAT/γPNA hairpin (self-cyclized TAT peptide) enters cells at least 10-fold more efficiently than its nonhairpin analog in which the two γPNA segments are noncomplementary. Extending one of the γPNA segments in the hairpin results in an overhang that can be used for binding and delivering a variety of nucleic acid-conjugated molecules into cells via hybridization to the overhang. We demonstrated efficient cellular delivery of a protein (as low as 10 nM) and a DNA tetrahedron by a TAT/γPNA hairpin.

Accessibility of Densely Localized DNA on Soft Polymer Nanoparticles.

The dense localization of DNA on soluble nanoparticles can lead to effects distinct from equivalent amounts of the DNA in solution. However, the specific effect may depend on the nature of the assembly and the nanoparticle core. Here we examine the accessibility of densely packed DNA duplexes that extend from a bottle-brush polymer core. We find that unlike spherical nucleic acids, the DNA duplex bristles on the bottle-brush polymer remain accessible to sequence-specific cleavage by endonucleases. In addition, the hybridized strand of the duplex can be displaced through a toehold-mediated strand exchange even at the polymer interface. These results demonstrate that the DNA on bottle-brush polymer remains sufficiently flexible to allow enzymatic degradation or DNA hybridization.

γPNA FRET Pair Miniprobes for Quantitative Fluorescent In Situ Hybridization to Telomeric DNA in Cells and Tissue.

Measurement of telomere length by fluorescent in situ hybridization is widely used for biomedical and epidemiological research, but there has been relatively little development of the technology in the 20 years since it was first reported. This report describes the use of dual gammaPNA (γPNA) probes that hybridize at alternating sites along a telomere and give rise to Förster resonance energy transfer (FRET) signals. Bright staining of telomeres is observed in nuclei, chromosome spreads and tissue samples. The use of FRET detection also allows for elimination of wash steps, normally required to remove unhybridized probes that would contribute to background signals. We found that these wash steps can diminish the signal intensity through the removal of bound, as well as unbound probes, so eliminating these steps not only accelerates the process but also enhances the quality of staining. Thus, γPNA FRET pairs allow for brighter and faster staining of telomeres in a wide range of research and clinical formats.

Fluoromodules Consisting of a Promiscuous RNA Aptamer and Red or Blue Fluorogenic Cyanine Dyes: Selection, Characterization, and Bioimaging.

An RNA aptamer selected for binding to the fluorogenic cyanine dye, dimethylindole red (DIR), also binds and activates another cyanine, oxazole thiazole blue (OTB), giving two well-resolved emission colors. The aptamer binds to each dye with submicromolar KD values, and the resulting fluoromodules exhibit fluorescence quantum yields ranging from 0.17 to 0.51 and excellent photostability. The aptamer was fused to a second aptamer previously selected for binding to the epidermal growth factor receptor (EGFR) to create a bifunctional aptamer that labels cell-surface EGFR on mammalian cells. The fluorescent color of the aptamer-labeled EGFR can be switched between blue and red in situ simply by exchanging the dye in the medium. The promiscuity of the aptamer can also be used to distinguish between cell-surface and internalized EGFR on the basis of the addition of red or blue fluorogen at different times.

RNA G-Quadruplex Invasion and Translation Inhibition by Antisense γ-Peptide Nucleic Acid Oligomers.

We have examined the abilities of three complementary γ-peptide nucleic acid (γPNA) oligomers to invade an RNA G-quadruplex and potently inhibit translation of a luciferase reporter transcript containing the quadruplex-forming sequence (QFS) within its 5'-untranslated region. All three γPNA oligomers bind with low nanomolar affinities to an RNA oligonucleotide containing the QFS. However, while all probes inhibit translation with low to midnanomolar IC50 values, the γPNA designed to hybridize to the first two G-tracts of the QFS and adjacent 5'-overhanging nucleotides was 5-6 times more potent than probes directed to either the 3'-end or internal regions of the target at 37 °C. This position-dependent effect was eliminated after the probes and target were preincubated at an elevated temperature prior to translation, demonstrating that kinetic effects exert significant control over quadruplex invasion and translation inhibition. We also found that antisense γPNAs exhibited similarly potent effects against luciferase reporter transcripts bearing QFS motifs having G2, G3, or G4 tracts. Finally, our results indicate that γPNA oligomers exhibit selectivity and/or potency higher than those of other antisense molecules such as standard PNA and 2'-OMe RNA previously reported to target G-quadruplexes in RNA.

Homologous PNA Hybridization to Noncanonical DNA G-Quadruplexes.

Potential guanine (G) quadruplex-forming sequences (QFSs) found throughout the genomes and transcriptomes of organisms have emerged as biologically relevant structures. These G-quadruplexes represent novel opportunities for gene regulation at the DNA and RNA levels. Recently, the definition of functional QFSs has been expanding to include a variety of unconventional motifs, including relatively long loop sequences (i.e., >7 nucleotides) separating adjacent G-tracts. We have identified a QFS within the 25S rDNA gene from Saccharomyces cerevisae that features a long loop separating the two 3'-most G-tracts. An oligonucleotide based on this sequence, QFS3, folds into a stable G-quadruplex in vitro. We have studied the interaction between QFS3 and several loop mutants with a small, homologous (G-rich) peptide nucleic acid (PNA) oligomer that is designed to form a DNA/PNA heteroquadruplex. The PNA successfully invades the DNA quadruplex target to form a stable heteroquadruplex, but with surprisingly high PNA:DNA ratios based on surface plasmon resonance and mass spectrometric results. A model for high stoichiometry PNA-DNA heteroquadruplexes is proposed, and the implications for quadruplex targeting by G-rich PNA are discussed.

Probing of miniPEGγ-PNA-DNA Hybrid Duplex Stability with AFM Force Spectroscopy.

Peptide nucleic acids (PNA) are synthetic polymers, the neutral peptide backbone of which provides elevated stability to PNA-PNA and PNA-DNA hybrid duplexes. It was demonstrated that incorporation of diethylene glycol (miniPEG) at the γ position of the peptide backbone increased the thermal stability of the hybrid duplexes (Sahu, B. et al. J. Org. Chem. 2011, 76, 5614-5627). Here, we applied atomic force microscopy (AFM) based single molecule force spectroscopy and dynamic force spectroscopy (DFS) to test the strength and stability of the hybrid 10 bp duplex. This hybrid duplex consisted of miniPEGγ-PNA and DNA of the same length (γ(MP)PNA-DNA), which we compared to a DNA duplex with a homologous sequence. AFM force spectroscopy data obtained at the same conditions showed that the γ(MP)PNA-DNA hybrid is more stable than the DNA counterpart, 65 ± 15 pN vs 47 ± 15 pN, respectively. The DFS measurements performed in a range of pulling speeds analyzed in the framework of the Bell-Evans approach yielded a dissociation constant, koff ≈ 0.030 ± 0.01 s⁻¹ for γ(MP)PNA-DNA hybrid duplex vs 0.375 ± 0.18 s⁻¹ for the DNA-DNA duplex suggesting that the hybrid duplex is much more stable. Correlating the high affinity of γ(MP)PNA-DNA to slow dissociation kinetics is consistent with prior bulk characterization by surface plasmon resonance. Given the growing interest in γ(MP)PNA as well as other synthetic DNA analogues, the use of single molecule experiments along with computational analysis of force spectroscopy data will provide direct characterization of various modifications as well as higher order structures such as triplexes and quadruplexes.

In Vitro Reversible Translation Control Using γPNA Probes.

On-demand regulation of gene expression in living cells is a central goal of chemical biology and antisense therapeutic development. While significant advances have allowed regulatory modulation through inserted genetic elements, on-demand control of the expression/translation state of a given native gene by complementary sequence interactions remains a technical challenge. Toward this objective, we demonstrate the reversible suppression of a luciferase gene in cell-free translation using Watson-Crick base pairing between the mRNA and a complementary γ-modified peptide nucleic acid (γPNA) sequence with a noncomplementary toehold. Exploiting the favorable thermodynamics of γPNA-γPNA interactions, the antisense sequence can be removed by hybridization of a second, fully complementary γPNA, through a strand displacement reaction, allowing translation to proceed. Complementary RNA is also shown to displace the bound antisense γPNA, opening up possibilities of in vivo regulation by native gene expression.

Spectral fine tuning of cyanine dyes: electron donor-acceptor substituted analogues of thiazole orange.

The introduction of electron donor and acceptor groups at strategic locations on a fluorogenic cyanine dye allows fine-tuning of the absorption and emission spectra while preserving the ability of the dye to bind to biomolecular hosts such as double-stranded DNA and a single-chain antibody fragment originally selected for binding to the parent unsubstituted dye, thiazole orange (TO). The observed spectral shifts are consistent with calculated HOMO-LUMO energy gaps and reflect electron density localization on the quinoline half of TO in the LUMO. A dye bearing donating methoxy and withdrawing trifluoromethyl groups on the benzothiazole and quinoline rings, respectively, shifts the absorption spectrum to sufficiently longer wavelengths to allow excitation at green wavelengths as opposed to the parent dye, which is optimally excited in the blue.

Bichromophoric dyes for wavelength shifting of dye-protein fluoromodules.

Dye-protein fluoromodules consist of fluorogenic dyes and single chain antibody fragments that form brightly fluorescent noncovalent complexes. This report describes two new bichromophoric dyes that extend the range of wavelengths of excitation or emission of existing fluoromodules. In one case, a fluorogenic thiazole orange (TO) was attached to an energy acceptor dye, Cy5. Upon binding to a protein that recognizes TO, red emission due to efficient energy transfer from TO to Cy5 replaces the green emission observed for monochromophoric TO bound to the same protein. Separately, TO was attached to a coumarin that serves as an energy donor. The same green emission is observed for coumarin-TO and TO bound to a protein, but efficient energy transfer allows violet excitation of coumarin-TO, versus longer wavelength, blue excitation of monochromophoric TO. Both bichromophores exhibit low nanomolar KD values for their respective proteins, >95% energy transfer efficiency and high fluorescence quantum yields.

Bright Fluorescent Nanotags from Bottlebrush Polymers with DNA-Tipped Bristles.

Bright signal outputs are needed for fluorescence detection of biomolecules at their native expression levels. Increasing the number of labels on a probe often results in crowding-induced self-quenching of chromophores, and maintaining the function of the targeting moiety (e.g., an antibody) is a concern. Here we demonstrate a simple method to accommodate thousands of fluorescent dye molecules on a single antibody probe while avoiding the negative effects of self-quenching. We use a bottlebrush polymer from which extend hundreds of duplex DNA strands that can accommodate hundreds of covalently attached and/or thousands of noncovalently intercalated fluorescent dyes. This polymer-DNA assembly sequesters the intercalated fluorophores against dissociation and can be tethered through DNA hybridization to an IgG antibody. The resulting fluorescent nanotag can detect protein targets in flow cytometry, confocal fluorescence microscopy, and dot blots with an exceptionally bright signal that compares favorably to commercially available antibodies labeled with organic dyes or quantum dots.