DNA Strand Displacement Driven by Host-Guest Interactions.

Base-pair-driven toehold-mediated strand displacement (BP-TMSD) is a fundamental concept employed for constructing DNA machines and networks with a gamut of applications─from theranostics to computational devices. To broaden the toolbox of dynamic DNA chemistry, herein, we introduce a synthetic surrogate termed host-guest-driven toehold-mediated strand displacement (HG-TMSD) that utilizes bioorthogonal, cucurbit[7]uril (CB[7]) interactions with guest-linked input sequences. Since control of the strand-displacement process is salient, we demonstrate how HG-TMSD can be finely modulated via changes to the structure of the input sequence (including synthetic guest head-group and/or linker length). Further, for a given input sequence, competing small-molecule guests can serve as effective regulators (with fine and coarse control) of HG-TMSD. To show integration into functional devices, we have incorporated HG-TMSD into machines that control enzyme activity and layered reactions that detect specific microRNA.

Structure/Function Analysis of Truncated Amino-Terminal ACE2 Peptide Analogs That Bind to SARS-CoV-2 Spike Glycoprotein.

The global burden of the SARS-CoV-2 pandemic is thought to result from a high viral transmission rate. Here, we consider mechanisms that influence host cell-virus binding between the SARS-CoV-2 spike glycoprotein (SPG) and the human angiotensin-converting enzyme 2 (ACE2) with a series of peptides designed to mimic key ACE2 hot spots through adopting a helical conformation analogous to the N-terminal α1 helix of ACE2, the region experimentally shown to bind to the SARS-CoV-2 receptor-binding domain (RBD). The approach examines putative structure/function relations by assessing SPG binding affinity with surface plasmon resonance (SPR). A cyclic peptide (c[KFNHEAEDLFEKLM]) was characterized in an α-helical conformation with micromolar affinity (KD = 500 µM) to the SPG. Thus, stabilizing the helical structure of the 14-mer through cyclization improves binding to SPG by an order of magnitude. In addition, end-group peptide analog modifications and residue substitutions mediate SPG binding, with net charge playing an apparent role. Therefore, we surveyed reported viral variants, and a correlation of increased positive charge with increased virulence lends support to the hypothesis that charge is relevant to enhanced viral fusion. Overall, the structure/function relationship informs the importance of conformation and charge for virus-binding analog design.

Chaperone-Assisted Host-Guest Interactions Revealed by Single-Molecule Force Spectroscopy.

The recent discovery of ultra-high binding affinities in cucurbit[7]uril (CB7)-based host-guest pairs in an aqueous environment has rendered CB7 a rather attractive material in analytical and biomedical applications. Due to the lack of a molecular platform that can follow the same host-guest complex during repetitive mechanical measurements, however, mechanical stabilities of the CB7 system have not been revealed, hindering its potential to rival widely used conjugation pairs, such as streptavidin-biotin. Here, we assembled a DNA template in which a flexible DNA linker was exploited to keep the host (CB7) and guest (adamantane) in proximity. This platform not only increased the efficiency of the single-molecule characterization in optical tweezers but also clearly revealed mechanical features of the same host-guest complex. We found that positively charged adamantane guest demonstrated higher mechanical stability (49 pN) than neutral adamantane (44 pN), a trend consistent with the chemical affinity between guest molecules and the CB7 host. Surprisingly, we found that a hexyl group adjacent to the adamantane served as a chaperone to assist the formation of the adamantane-CB7 pairs. The discovery of an unprecedented chaperone-assisted interaction mechanism provides new approaches to efficiently assemble host-guest-based supramolecules with increased mechanical stabilities, which can be exploited in various biomedical, biosensing, and materials fields.

Bright G-Quadruplex Nanostructures Functionalized with Porphyrin Lanterns.

The intricate arrangement of numerous and closely placed chromophores on nanoscale scaffolds can lead to key photonic applications ranging from optical waveguides and antennas to signal-enhanced fluorescent sensors. In this regard, the self-assembly of dye-appended DNA sequences into programmed photonic architectures is promising. However, the dense packing of dyes can result in not only compromised DNA assembly (leading to ill-defined structures and precipitates) but also to essentially nonfluorescent systems (due to π-π aggregation). Here, we introduce a two-step "tether and mask" strategy wherein large porphyrin dyes are first attached to short G-quadruplex-forming sequences and then reacted with per-O-methylated ß-cyclodextrin (PMßCD) caps, to form supramolecular synthons featuring the porphyrin fluor fixed into a masked porphyrin lantern (PL) state, due to intramolecular host-guest interactions in water. The PL-DNA sequences can then be self-assembled into cyclic architectures or unprecedented G-wires tethered with hundreds of porphyrin dyes. Importantly, despite the closely arrayed PL units (∼2 nm), the dyes behave as bright chromophores (up to 180-fold brighter than the analogues lacking the PMßCD masks). Since other self-assembling scaffolds, dyes, and host molecules can be used in this modular approach, this work lays out a general strategy for the bottom-up aqueous self-assembly of bright nanomaterials containing densely packed dyes.

Host-Guest Tethered DNA Transducer: ATP Fueled Release of a Protein Inhibitor from Cucurbit[7]uril.

Host-guest complexes are emerging as powerful components in functional systems with applications ranging from materials to biomedicine. In particular, CB7 based host-guest complexes have received much attention for the controlled release of drugs due to the remarkable ability of CB7 toward binding input molecules in water with high affinity leading to displacement of CB7 from included pharmacophores (or from drug loaded porous particles). However, the release of bound guests from CB7 in response to endogenous biological molecules remains limited since the input biomolecule needs to have the appropriate chemical structure to bind tightly into the CB7 cavity. Herein we describe a synthetic transducer based on self-assembling DNA-small molecule chimeras (DCs) that is capable of converting a chosen biological input, adenosine triphosphate (ATP; that does not directly bind to the CB7 host), into functional displacement of a protein inhibitor that is bound within the CB7 host. Our system-which features the first example of a covalent CB-DNA conjugate-is highly modular and can be adapted to enable responsiveness to other biologically/clinically relevant stimuli via its split DNA aptamer architecture.

Elevated expression of long intergenic non-coding RNA HOTAIR in a basal-like variant of MCF-7 breast cancer cells.

Epigenetic regulation of gene expression is critical to phenotypic maintenance and transition of human breast cancer cells. HOX antisense intergenic RNA (HOTAIR) is a long intergenic non-coding RNA that epigenetically represses gene expression via recruitment of enhancer of zeste homolog 2 (EZH2), a histone methyltransferase. Elevated expression of HOTAIR promotes progression of breast cancer. In the current study we examined the expression and function of HOTAIR in MCF-7-TNR cells, a derivative of the luminal-like breast cancer cell line MCF-7 that acquired resistance to TNF-α-induced cell death. The expression of HOTAIR, markers of the luminal-like and basal-like subtypes, and growth were compared between MCF-7 and MCF-7-TNR cells. These variables were further assessed upon inhibition of HOTAIR, EZH2, p38 MAPK, and SRC kinase in MCF-7-TNR cells. When compared with MCF-7 cells, MCF-7-TNR cells exhibited an increase in the expression of HOTAIR, which correlated with characteristics of a luminal-like to basal-like transition as evidenced by dysregulated gene expression and accelerated growth. MCF-7-TNR cells exhibited reduced suppressive histone H3 lysine27 trimethylation on the HOTAIR promoter. Inhibition of HOTAIR and EZH2 attenuated the luminal-like to basal-like transition in terms of gene expression and growth in MCF-7-TNR cells. Inhibition of p38 and SRC diminished HOTAIR expression and the basal-like phenotype in MCF-7-TNR cells. HOTAIR was robustly expressed in the native basal-like breast cancer cells and inhibition of HOTAIR reduced the basal-like gene expression and growth. Our findings suggest HOTAIR-mediated regulation of gene expression and growth associated with the basal-like phenotype of breast cancer cells.

Bile Acid Conjugated DNA Chimera that Conditionally Inhibits Carbonic Anhydrase-II in the Presence of MicroRNA-21.

In order to tackle the issue of systemic toxicity in chemotherapy, there is a need to develop novel mechanisms for the activation of protein inhibitors using biomarkers overexpressed in cancer cells. Many current strategies focus on using cancer associated enzymes as a triggering agent for prodrugs. Herein, we detail an alternative approach that harnesses a microRNA (miR-21) that is overexpressed in cancers as the trigger that activates an inhibitor of human carbonic anhydrase-II (hCA-II). Specifically, we have developed a DNA-small molecule chimera (DC) composed of an hCA-II binding lithocholic acid amide (LAA) headgroup that can transition from a rigid duplex state (that does not bind appreciably to hCA) to a single-stranded conformation via a miR-21 trigger. The activated single-stranded DC can project the LAA headgroup into the hCA-II active site and is a robust hCA-II inhibitor (K(i) of 3.12 µM). This work may spur research into developing new classes of cancer selective protein inhibitors.

Host-guest interactions derived multilayer perylene diimide thin film constructed on a scaffolding porphyrin monolayer.

The development of methods to grow well-ordered chromophore thin films on solid substrates is of importance because such surface-associated arrays have potential applications in the generation of functional electronic and optical materials and devices. In this article, we demonstrate a straightforward layer-by-layer (LBL) supramolecular deposition strategy to prepare numerous layers (up to 19) of functionalized perylene diimide (PDI) chromophores built upon a covalent scaffolding multivalent porphyrin monolayer. Our thin film formation strategy employs water as the immersion solvent and exploits the ß-cyclodextrin-adamantane host-guest couple in addition to PDI based aromatic stacking. Within the resultant film the porphyrin scaffold is oriented close to parallel to the glass substrate while the PDI chromophores are aligned closer to the surface normal. Together, the porphyrin monolayer and the multi-PDI layers exhibit a large absorption bandwidth in the visible spectrum. Importantly, because a self-assembly strategy was utilized, when a single monolayer of PDI is deposited on the porphyrin scaffolding layer, this PDI monolayer can be readily disassembled by washing with DMF leading to the regeneration of the porphyrin monolayer. The PDI thin film can subsequently be regrown from the regenerated porphyrin surface. The reported LBL strategy will be of broad interest for researchers developing well-organized chromophoric films and materials due to its simplicity as well as the added advantage of being performed in sustainable and cost-effective aqueous media.

Room-temperature ballistic energy transport in molecules with repeating units.

In materials, energy can propagate by means of two limiting regimes: diffusive and ballistic. Ballistic energy transport can be fast and efficient and often occurs with a constant speed. Using two-dimensional infrared spectroscopy methods, we discovered ballistic energy transport via individual polyethylene chains with a remarkably high speed of 1440 m/s and the mean free path length of 14.6 Å in solution at room temperature. Whereas the transport via the chains occurs ballistically, the mechanism switches to diffusive with the effective transport speed of 130 m/s at the end-groups attached to the chains. A unifying model of the transport in molecules is presented with clear time separation and additivity among the transport along oligomeric fragments, which occurs ballistically, and the transport within the disordered fragments, occurring diffusively. The results open new avenues for making novel elements for molecular electronics, including ultrafast energy transporters, controlled chemical reactors, and sub-wavelength quantum nanoseparators.

Mild two-step method to construct DNA-conjugated silicon nanoparticles: scaffolds for the detection of microRNA-21.

We describe a novel two-step method, starting from bulk silicon wafers, to construct DNA conjugated silicon nanoparticles (SiNPs). This method first utilizes reactive high-energy ball milling (RHEBM) to obtain alkene grafted SiNPs. The alkene moieties are subsequently reacted with commercially available thiol-functionalized DNA via thiol-ene click chemistry to produce SiNP DNA conjugates wherein the DNA is attached through a covalent thioether bond. Further, to show the utility of this synthetic strategy, we illustrate how these SiNP ODN conjugates can detect cancer-associated miR-21 via a fluorescence ON strategy. Given that an array of biological molecules can be prepared with thiol termini and that SiNPs are biocompatible and biodegradable, we envision that this synthetic protocol will find utility in salient SiNP systems for potential therapeutic and diagnostic applications.

Photonic DNA-chromophore nanowire networks: harnessing multiple supramolecular assembly modes.

Photonic DNA nanostructures are typically prepared by the assembly of multiple sequences of long DNA strands that are conjugated covalently to various dye molecules. Herein we introduce a noncovalent method for the construction of porphyrin-containing DNA nanowires and their networks that uses the programmed assembly of a single, very short, oligodeoxyribonucleotide sequence. Specifically, our strategy exploits a number of supramolecular binding modalities (including DNA base-pairing, metal-ion coordination, and ß-cyclodextrin-adamantane derived host-guest interactions) for simultaneous nanowire assembly and porphyrin incorporation. Furthermore, we also show that the resultant DNA-porphyrin assembly can be further functionalized with a complementary "off-the-shelf" DNA binding dye resulting in photonic structures with broadband absorption and energy transfer capabilities.

Oligonucleotide-Based Systems for Input-Controlled and Non-Covalently Regulated Protein-Binding.

Supramolecular chemists continuously take inspiration from complex biological systems to develop functional molecules involved in molecular recognition and self-assembly. In this regard, "smart" synthetic molecules that emulate allosteric proteins are both exciting and challenging, since many allosteric proteins can be considered as molecular switches that bind to other protein targets in a non-covalent fashion, and importantly, are capable of having their output activity controlled by prior binding to input molecules. This review discusses the foundations and passage toward the development of non-covalently operated oligonucleotide-based systems with protein-binding capacity that can be precisely regulated in an input-controlled manner.

Heteromeric guanosine (G)-quadruplex derived antenna modules with directional energy transfer.

A heteromeric guanosine (G)-quadruplex centered self-assembly approach is developed to prepare compact light-harvesting antenna modules featuring multiple donor dyes and a single toehold region. Due to the mix-and-match nature of our approach, the number and placement of donor dyes can be readily fine-tuned via quadruplex assembly. Moreover, hybridization of the toehold with an acceptor containing sequence results in directional energy transfer ensembles with effective absorption coefficients in the 105 M-1 cm-1 range. These compact antennas exhibit system efficiencies that are comparable to much larger and elaborate DNA architectures containing numerous DNA strands.

G-quadruplex is critical to epigenetic activation of the lncRNA HOTAIR in cancer cells.

The cancer-promoting lncRNA HOTAIR has multiple isoforms. Which isoform of HOTAIR accounts for its expression and functions in cancer is unknown. Unlike HOTAIR's canonical intergenic isoform NR_003716 (HOTAIR-C), the novel isoform NR_047517 (HOTAIR-N) forms an overlapping antisense transcription locus with HOXC11. We identified HOTAIR-N as the dominant isoform that regulates the gene expression programs and networks for cell proliferation, survival, and death in cancer cells. The CpG island in the HOTAIR-N promoter was marked with epigenetic markers for active transcription. We identified a G-quadruplex (G4) motif rich region in the HOTAIR-N CpG island. Our findings indicate that G4s in HOTAIR-N CpG island is critical for expression of HOTAIR-N in cancer cells. Disruption of G4 may represent a novel therapeutic approach for cancer. The transcriptomes regulated by HOTAIR-N and Bloom in cancer cells as provided herein are important resources for the exploration of lncRNA, DNA helicases, and G4 in cancer.

Ballistic energy transport along PEG chains: distance dependence of the transport efficiency.

Dual-frequency relaxation-assisted two-dimensional infrared (RA 2DIR) spectroscopy was used to investigate energy transport in polyethylene glycol (PEG) oligomers of different length, having 0, 4, 8, and 12 repeating units and end-labeled with azido and succinimide ester moieties (azPEGn). The energy transport initiated by excitation of the N≡N stretching mode of the azido group in azPEGn in CCl(4) at ca. 2100 cm(-1) was recorded by probing the C=O stretching modes (reporters) of the succinimide ester moiety. Sensitive to the excess energy delivered to the reporter modes, RA 2DIR permits observation of both the through-bond and through-solvent energy transport contributions. The cross-peak data involving the reporter modes with different thermal sensitivity and the data for mixtures of compounds permitted concluding that through-bond energy transport is the dominant mechanism for most cross peaks in all four azPEGn compounds. The through-bond energy transport time, evaluated as the waiting time at which the cross peak maximum is reached, was found to be linearly dependent on the chain length of up to 60 Å, suggesting a ballistic energy transport regime. The through-bond energy transport speed determined from the chain-length dependence of T(max) in CCl(4) is found to be ca. 450 m s(-1). The cross-peak amplitude at the maximum decays exponentially with the chain length; a characteristic decay distance is found to be 15.7 ± 1 Å. The cross-peak amplitude at zero waiting time, determined by the end-to-end distance distribution, is found to decay with the chain length (L) as ∼L(-1.4), which is close to predictions of the free flight chain model. The match indicates that the end-group interaction does not strongly perturb the end-to-end distribution, which is close to the ideal random coil distribution with the Gaussian probability density.

A combination of novel NSC small molecule inhibitor along with doxorubicin inhibits proliferation of triple-negative breast cancer through metabolic reprogramming.

Treatment of patients with triple-negative breast cancer (TNBC) has been challenging due to the absence of well-defined molecular targets and the highly invasive and proliferative nature of TNBC cells. Current treatments against TNBC have shown little promise due to high recurrence rate in patients. Consequently, there is a pressing need for novel and efficacious therapies against TNBC. Here, we report the discovery of a novel small molecule inhibitor (NSC33353) with potent anti-tumor activity against TNBC cells. The anti-proliferative effects of this small molecule inhibitor were determined using 2D and 3D cell proliferation assays. We found that NSC33353 significantly reduces the proliferation of TNBC cells in these assays. Using proteomics, next generation sequencing (NGS), and gene enrichment analysis, we investigated global regulatory pathways affected by this compound in TNBC cells. Proteomics data indicate a significant metabolic reprograming affecting both glycolytic enzymes and energy generation through oxidative phosphorylation. Subsequently, using metabolic (Seahorse) and enzymatic assays, we validated our proteomics and NGS analysis findings. Finally, we showed the inhibitory and anti-tumor effects of this small molecule in vitro and confirmed its inhibitory activity in vivo. Doxorubicin is one of the most effective agents in the treatment of TNBC and resistance to this drug has been a major problem. We show that the combination of NSC33353 and doxorubicin suppresses the growth of TNBC cells synergistically, suggesting that NSC33353 enhances TNBC sensitivity to doxorubicin. In summary, our data indicate that the small molecule inhibitor, NSC33353, exhibits anti-tumor activity in TNBC cells, and works in a synergistic fashion with a well-known chemotherapeutic agent.

Protein-binding molecular switches via host-guest stabilized DNA hairpins.

Molecular switches, with target protein-binding activity controlled by prior binding to specific input stimuli, are ubiquitously used in Nature. However, the emulation of such responsive systems, especially in a de novo fashion, remains a significant challenge. Herein, we disclose a strategy that harnesses an intramolecular ß-CD/adamantane host-guest interaction to generate a stabilized DNA hairpin (ΔT(m) = 17 °C) that undergoes an input oligonucleotide (ODN)-selective structural transformation from a stem-loop conformation to a duplex. This ODN-induced conformational switch allows for the transition from an inactive state (wherein the adamantane protein-binding headgroup is encapsulated) to an activated protein-binding complex, with a freely accessible adamantane moiety. Given that hairpin domains can be readily modulated to be responsive to alternative ODN triggering sequences and that encapsulating macrocycles, such as ß-CD, are good hosts for a number of protein-binding small molecules, this strategy may furnish a general method to develop ODN-responsive protein-binders.

Molecular recognition and enhancement of aqueous solubility and bioactivity of CD437 by ß-cyclodextrin.

CD437 (6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid) is a novel synthetic retinoic acid derivative that has been shown to selectively induce apoptosis in human lung cancer cells. This compound, however, is limited in its application due to its low solubility in aqueous solutions. One technique for increasing the solubility and bioavailability of a cytotoxic agent is the formation of inclusion complexes with cyclodextrins. Herein, we report the formation and characterization of a 2:1 complex between ß-cyclodextrin (ß-CD) and CD437. It is shown that CD437 is a tight binder of ß-CD with an overall association constant of 2.6±0.6×10(7)M(-2). In addition, we demonstrate (a) that ß-CD-derived complexation enhances the aqueous solubility of CD437, and (b) that a significant increase in the toxicity of CD437 against a human lung adenocarcinoma cell line can be achieved by co-treatment with ß-CD.

Synthesis and Applications of Porphyrin-Biomacromolecule Conjugates.

With potential applications in materials and especially in light-responsive biomedicine that targets cancer tissue selectively, much research has focused on developing covalent conjugation techniques to tether porphyrinoid units to various biomacromolecules. This review details the key synthetic approaches that have been employed in the recent decades to conjugate porphyrinoids with oligonucleotides and peptides/proteins. In addition, we provide succinct discussions on the subsequent applications of such hybrid systems and also give a brief overview of the rapidly progressing field of porphyrin-antibody conjugates. Since nucleic acid and peptide systems vary in structure, connectivity, functional group availability and placement, as well as stability and solubility, tailored synthetic approaches are needed for conjugating to each of these biomacromolecule types. In terms of tethering to ONs, porphyrins are typically attached by employing bioorthogonal chemistry (e.g., using phosphoramidites) that drive solid-phase ON synthesis or by conducting post-synthesis modifications and subsequent reactions (such as amide couplings, hydrazide-carbonyl reactions, and click chemistry). In contrast, peptides and proteins are typically conjugated to porphyrinoids using their native functional groups, especially the thiol and amine side chains. However, bioorthogonal reactions (e.g., Staudinger ligations, and copper or strain promoted alkyne-azide cycloadditions) that utilize de novo introduced functional groups onto peptides/proteins have seen vigorous development, especially for site-specific peptide-porphyrin tethering. While the ON-porphyrin conjugates have largely been explored for programmed nanostructure self-assembly and artificial light-harvesting applications, there are some reports of ON-porphyrin systems targeting clinically translational applications (e.g., antimicrobial biomaterials and site-specific nucleic acid cleavage). Conjugates of porphyrins with proteinaceous moieties, on the other hand, have been predominantly used for therapeutic and diagnostic applications (especially in photodynamic therapy, photodynamic antimicrobial chemotherapy, and photothermal therapy). The advancement of the field of porphyrinoid-bioconjugation chemistry from basic academic research to more clinically targeted applications require continuous fine-tuning in terms of synthetic strategies and hence there will continue to be much exciting work on porphyrinoid-biomacromolecule conjugation.