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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Modular evolution of DNA-binding preference of a Tbrain transcription factor provides a mechanism for modifying gene regulatory networks.

Gene regulatory networks (GRNs) describe the progression of transcriptional states that take a single-celled zygote to a multicellular organism. It is well documented that GRNs can evolve extensively through mutations to cis-regulatory modules (CRMs). Transcription factor proteins that bind these CRMs may also evolve to produce novelty. Coding changes are considered to be rarer, however, because transcription factors are multifunctional and hence are more constrained to evolve in ways that will not produce widespread detrimental effects. Recent technological advances have unearthed a surprising variation in DNA-binding abilities, such that individual transcription factors may recognize both a preferred primary motif and an additional secondary motif. This provides a source of modularity in function. Here, we demonstrate that orthologous transcription factors can also evolve a changed preference for a secondary binding motif, thereby offering an unexplored mechanism for GRN evolution. Using protein-binding microarray, surface plasmon resonance, and in vivo reporter assays, we demonstrate an important difference in DNA-binding preference between Tbrain protein orthologs in two species of echinoderms, the sea star, Patiria miniata, and the sea urchin, Strongylocentrotus purpuratus. Although both orthologs recognize the same primary motif, only the sea star Tbr also has a secondary binding motif. Our in vivo assays demonstrate that this difference may allow for greater evolutionary change in timing of regulatory control. This uncovers a layer of transcription factor binding divergence that could exist for many pairs of orthologs. We hypothesize that this divergence provides modularity that allows orthologous transcription factors to evolve novel roles in GRNs through modification of binding to secondary sites.

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.

Hybridization of G-quadruplex-forming peptide nucleic acids to guanine-rich DNA templates inhibits DNA polymerase η extension.

The guanine quadruplex (G-quadruplex) is a highly stable secondary structure that forms in G-rich repeats of DNA, which can interfere with DNA processes, including DNA replication and transcription. We showed previously that short guanine-rich peptide nucleic acids (PNAs) can form highly stable hybrid quadruplexes with DNA. We hypothesized that such structures would provide a stronger block to polymerase extension on G-rich templates than a native DNA homoquadruplex because of the greater thermodynamic stability of the PNA-DNA hybrid structures. To test this, we analyzed the DNA primer extension activity of polymerase η, a translesion polymerase implicated in synthesis past G-quadruplex blocks, on DNA templates containing guanine repeats. We observed a PNA concentration-dependent decrease in the level of polymerase η extension to the end of the template and an increase in the level of polymerase η inhibition at the sequence prior to the G-rich repeats. In contrast, the addition of a complementary C-rich PNA that hybridizes to the G-rich repeats by Watson-Crick base pairing led to a decrease in the level of polymerase inhibition and an increase in the level of full-length extension products. The G-quadruplex-forming PNA exhibited inhibition (IC50=16.2±3.3 nM) of polymerase η DNA synthesis on the G-rich templates stronger than that of the established G-quadruplex-stabilizing ligand BRACO-19 (IC50=42.5±4.8 nM). Our results indicate that homologous PNA targeting of G-rich sequences creates stable PNA-DNA heteroquadruplexes that inhibit polymerase η extension more effectively than a DNA homoquadruplex. The implications of these results for the potential development of homologous PNAs as therapeutics for halting proliferating cancer cells are discussed.

Label-free molecular beacons for biomolecular detection.

Biomolecular detection and imaging methods provide quantitative measurements essential for biological research. In this context, molecular beacon based sensors have emerged as powerful, no-wash imaging agents, providing target-specific fluorescent activation for nucleic acids, proteins, and small molecules. Conventional molecular beacons require double-labeled DNA sequences, which are costly and time-consuming to prepare. To address this issue, we developed DNA based label-free molecular beacons consisting of two regions: a signal-generating region based on human telomeric G-quadruplex sequence that activates Thioflavin T fluorescence and a target recognition sequence designed to interact in a molecular beacon format. We demonstrated the utility of these probes for the selective detection of DNA, RNA, and protein. Multiple probes were applied against a single target to achieve improved brightness in fluorescence detection of nucleic acid targets. This label-free strategy provides a straightforward, cost-effective alternative to fluorescently labeled oligonucleotides in biomolecular detection and imaging.

Aptamers act as activators for the thrombin mediated-hydrolysis of peptide substrates.

Thrombin is the typical target in anticlotting therapy for many serious diseases such as heart attack and stroke. DNA aptamers are well-known thrombin inhibitors that prevent fibrinogen hydrolysis. We have discovered that exosite-targeting antithrombin aptamers enhance the activity of thrombin toward a small peptide substrate, Sar(N-methylglycine)-Pro-Arg-paranitroanilide, and that the activation of the enzyme by these aptamers is strongly inhibited by their complementary DNAs. Our study reveals that treatment with mixed aptamers or with a dual-aptamer construct led to an 8.6- or 7.8-fold enhancement in peptide hydrolysis relative to thrombin alone, a synergistic effect much higher than the activation observed with a monofunctional aptamer (1.5-fold for Apt27 or 2.7-fold for Apt15). In addition, we discovered that Apt27 is a biofunctional molecule for thrombin because of its activation effect. An enzyme kinetic study indicates that the binding of aptamers to exosites I and II significantly activates thrombin towards the peptide substrate, thus illustrating that binding of aptamers to exosites can allosterically regulate the active site of thrombin. Our study suggests the necessity of considering possible side effects when DNA aptamers are used for clinical applications involving the inhibition of thrombin-mediated clotting.

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.