Athermal, Chemically Triggered Release of RNA from Thioester Nucleic Acids.

An athermal approach to mRNA enrichment from total RNA using a self-immolative thioester linked nucleic acids (TENA) is described. Oligo(thymine) (oT) TENA has a six-atom spacing between bases which allowed TENA to selectively base-pair with polyadenine RNA. As a result of the neutral backbone of TENA and the hydrophobicity of the octanethiol end group, oT TENA is water insoluble and efficiently pulled down 93±2 % of EGFP mRNA at a concentration of 10 ng µL-1 . Self-immolative degradation of TENA upon ambient temperature exposure to nucleophilic buffer components (Tris, DTT) allowed recovery of 55±27 ng of mRNA from 3.1 µg of total RNA, which was not statistically different from the amount recovered using Dynabeads® mRNA DIRECT Kit (89±24 ng). Gene expression as measured by RT-qPCR was comparable for both enrichment methods, suggesting that the mild conditions required for enrichment of mRNA using oT TENA are compatible with RT-qPCR and other downstream molecular biology applications.

Click Nucleic Acid-DNA Binding Behavior: Dependence on Length, Sequence, and Ionic Strength.

Click nucleic acids (CNAs) are a new, low-cost class of xeno nucleic acid (XNA) oligonucleotides synthesized by an efficient and scalable thiol-ene polymerization. In this work, a thorough characterization of oligo(thymine) CNA-oligo(adenine) DNA ((dA)20) hybridization was performed to guide the future implementation of CNAs in applications that rely on sequence-specific interactions. Microscale thermophoresis provided a convenient platform to rapidly and systematically investigate the effects of several factors (i.e., sequence, length, and salt concentration) on the CNA-DNA dissociation constant (Kapp). Because CNAs have limited water solubility, all studies were performed in aqueous-DMSO mixtures. CNA-DNA hybrids between oligo(thymine) CNA (average length of 16 bases) and (dA)20 DNA have good stability despite the high organic content, a favorable attribute for many emerging applications of XNAs. In particular, the Kapp of CNA-DNA hybrids in 65 vol % DMSO with 10 mM sodium chloride (NaCl) was 0.74 ± 0.1 µM, whereas the Kapp for (dT)20-(dA)20 DNA-DNA was found to be 45 ± 2 µM in a buffer without DMSO but at the same NaCl concentration. CNA hybridized with DNA following Watson-Crick base pairing with excellent sequence specificity, discriminating even a single-base-pair mismatch, with Kapp values of 0.74 ± 0.1 and 3.7 ± 0.6 µM for complementary and single-base-pair mismatch sequences, respectively. As with dsDNA, increasing CNA length led to more stable hybrids as a result of increased base pairing, where Kapp decreased from 5.6 ± 0.8 to 0.27 ± 0.1 µM as the CNA average length increased from 7 to 21 bases. However, unlike DNA-DNA duplexes, which are largely unstable at low salt concentrations, the CNA-DNA stability does not depend on salt concentration, with Kapp remaining consistent between 1.0 and1.9 µM over a NaCl concentration range of 1.25-30 mM.

Photo-responsive liposomes composed of spiropyran-containing triazole-phosphatidylcholine: investigation of merocyanine-stacking effects on liposome-fiber assembly-transition.

A spiropyran-containing triazole-phosphatidylcholine (SPTPC) was synthesized through a copper-catalyzed azide alkyne cyclo-addition (CuAAC) reaction. In water, SPTPCs self-assembled and a spontaneous spiropyran-to-merocyanine (SP-to-MC) isomerization occurred, resulting in coexistence of liposomes and fibers, and switching from the spiropyran (SP) to the merocyanine (MC) isomeric structure induced a reversible transition between these molecular assemblies. Study of the self-assembly of SPTPCs and photo-induced liposome-fiber assembly-transition revealed that the presence of MC enabled additional inter-membrane interaction during self-assembly and that the MC-stacking effect was the driving force for the assembly-transition. Exposure to UV light induced switching from SP to MC, where the planar structure of MC and the confinement of MC led to enhanced MC-stacking. The effect of MC-stacking was both advantageous and disadvantageous: MC-stacking perturbed the hydrophobic phase in the bilayer membrane and facilitated the liposome-to-fiber transition, otherwise the MC-stacking retarded switching of MC to SP, and caused an incomplete recovery of MC to SP during fiber-to-liposome recovery, thus a fatigue of SP was induced by MC-stacking during the liposome-to-fiber transition cycle. To decrease the intermolecular interactions and suppress MC-stacking, photo-inert triazole-phosphatidylcholine (TPC) was incorporated to prepare two-component TPC/SPTPC-liposomes, which exhibited better recovery kinetics. The photo-adaptive behavior of TPC/SPTPC-liposomes confirmed the disturbance of bilayer membranes by inter-membrane MC-stacking and the formation of MCTPC-enriched phases in the bilayer membrane.

Dynamic and Responsive DNA-like Polymers.

The synthesis of thiolactone monomers that mimic natural nucleosides and engage in robust ring opening polymerizations (ROP) is herein described. As each repeat unit contains a thioester functional group, dynamic rearrangement of the polymer is feasible via thiol-thioester exchange, demonstrated here by depolymerization of the polymers and coalescing of two polymers of different molecular weight or chemical composition. This approach constitutes the first step toward a platform that enables for the routine synthesis of sequence controlled polymers via dynamic template directed synthesis.

Analyte-Responsive Hydrogels: Intelligent Materials for Biosensing and Drug Delivery.

Nature has mastered the art of molecular recognition. For example, using synergistic non-covalent interactions, proteins can distinguish between molecules and bind a partner with incredible affinity and specificity. Scientists have developed, and continue to develop, techniques to investigate and better understand molecular recognition. As a consequence, analyte-responsive hydrogels that mimic these recognitive processes have emerged as a class of intelligent materials. These materials are unique not only in the type of analyte to which they respond but also in how molecular recognition is achieved and how the hydrogel responds to the analyte. Traditional intelligent hydrogels can respond to environmental cues such as pH, temperature, and ionic strength. The functional monomers used to make these hydrogels can be varied to achieve responsive behavior. For analyte-responsive hydrogels, molecular recognition can also be achieved by incorporating biomolecules with inherent molecular recognition properties (e.g., nucleic acids, peptides, enzymes, etc.) into the polymer network. Furthermore, in addition to typical swelling/syneresis responses, these materials exhibit unique responsive behaviors, such as gel assembly or disassembly, upon interaction with the target analyte. With the diverse tools available for molecular recognition and the ability to generate unique responsive behaviors, analyte-responsive hydrogels have found great utility in a wide range of applications. In this Account, we discuss strategies for making four different classes of analyte-responsive hydrogels, specifically, non-imprinted, molecularly imprinted, biomolecule-containing, and enzymatically responsive hydrogels. Then we explore how these materials have been incorporated into sensors and drug delivery systems, highlighting examples that demonstrate the versatility of these materials. For example, in addition to the molecular recognition properties of analyte-responsive hydrogels, the physicochemical changes that are induced upon analyte binding can be exploited to generate a detectable signal for sensing applications. As research in this area has grown, a number of creative approaches for improving the selectivity and sensitivity (i.e., detection limit) of these sensors have emerged. For applications in drug delivery systems, therapeutic release can be triggered by competitive molecular interactions or physicochemical changes in the network. Additionally, including degradable units within the network can enable sustained and responsive therapeutic release. Several exciting examples exploiting the analyte-responsive behavior of hydrogels for the treatment of cancer, diabetes, and irritable bowel syndrome are discussed in detail. We expect that creative and combinatorial approaches used in the design of analyte-responsive hydrogels will continue to yield materials with great potential in the fields of sensing and drug delivery.

New Generation of Clickable Nucleic Acids: Synthesis and Active Hybridization with DNA.

Due to the ability to generate oligomers of precise sequence, sequential and stepwise solid-phase synthesis has been the dominant method of producing DNA and other oligonucleotide analogues. The requirement for a solid support, however, and the physical restrictions of limited surface area thereon significantly diminish the efficiency and scalability of these syntheses, thus, negatively affecting the practical applications of synthetic polynucleotides and other similarly created molecules. By employing the robust photoinitiated thiol-ene click reaction, we developed a new generation of clickable nucleic acids (CNAs) with a polythioether backbone containing repeat units of six atoms, matching the spacing of the phosphodiester backbone of natural DNA. A simple, inexpensive, and scalable route was utilized to produce CNA monomers in gram-scale, which indicates the potential to dramatically lower the cost of these DNA mimics and thereby expand the scope of these materials. The efficiency of this approach was demonstrated by the completion of CNA polymerization in 30 seconds, as characterized by size-exclusive chromatography (SEC) and infrared (IR) spectroscopy. CNA/DNA hybridization was demonstrated by gel electrophoresis and used in CdS nanoparticle assembly.

Charged poly(N-isopropylacrylamide) nanogels for use as differential protein receptors in a turbidimetric sensor array.

Due to the high cost and environmental instability of antibodies, there is precedent for developing synthetic molecular recognition agents for use in diagnostic sensors. While these materials typically have lower specificity than antibodies, their cross-reactivity makes them excellent candidates for use in differential sensing routines. In the current work, we design a set of charge-containing poly(N-isopropylacrylamide) (PNIPAM) nanogels for use as differential protein receptors in a turbidimetric sensor array. Specifically, NIPAM was copolymerized with methacrylic acid and modified via carbodiimide coupling to introduce sulfate, guanidinium, secondary amine, or primary amine groups. Modification of the ionizable groups in the network changed the physicochemical and protein binding properties of the nanogels. For high affinity protein-polymer interactions, turbidity of the nanogel solution increased, while for low affinity interactions minimal change in turbidity was observed. Thus, relative turbidity was used as input for multivariate analysis. Turbidimetric assays were performed in two buffers of different pH (i.e., 7.4 and 5.5), but comparable ionic strength, in order to improve differentiation. Using both buffers, it was possible to achieve 100% classification accuracy of eleven model protein biomarkers with as few as two of the nanogel receptors. Additionally, it was possible to detect changes in lysozyme concentration in a simulated tear fluid using the turbidimetric sensor array.

Versatile Route to Colloidal Stability and Surface Functionalization of Hydrophobic Nanomaterials.

We introduce a general method for the stabilization and surface functionalization of hydrophobic nanoparticles using an amphiphilic copolymer, poly(maleic anhydride-alt-1-octadecene)-poly(ethylene glycol) methacrylate (PMAO-PEGMA). Coating nanoparticles with PMAO-PEGMA results in colloidally stable nanoparticles decorated with reactive carboxylic acid and methacrylate functionalities, providing a versatile platform for chemical reactions. The versatility and ease of surface functionalization is demonstrated by varying both the core material and the chemistry used. Specifically, the carboxylic acid functionalities are used to conjugate wheat germ agglutinin to conducting polymer nanoparticles via carbodiimide-mediated coupling, and the methacrylate groups are used to link cysteamine to the surface of poly(ε-caprolactone) nanoparticles via thiol-ene click chemistry and to link temperature-responsive polymer shells to the surface of gold nanoparticles via free radical polymerization.

A Closer Look at the Impact of Molecular Imprinting on Adsorption Capacity and Selectivity for Protein Templates.

Molecularly imprinted polymers (MIPs) are often investigated as lower cost, more environmentally robust alternatives to natural recognitive biomolecules, such as antibodies. When synthesized on the surface of nanomaterial supports, MIPs are capable of quick and effective binding of macromolecular templates when compared to traditional bulk-imprinted polymers. We have developed a method for imprinting proteins on biodegradable nanoparticle supports and have used these materials to investigate the impact of molecular imprinting on adsorption capacity and selectivity for lysozyme, the template protein. The imprinting process increased the adsorption capacity of the polymer for the template, lysozyme, with the MIPs being able to bind up to 83.5% of their dry weight as compared to 55.7% for nonimprinted polymers (NIPs). In noncompetitive binding experiments, where proteins were independently incubated with MIPs, the difference between adsorption capacity for lysozyme and proteins with much lower isoelectric points (pI < 8.0) was statistically significant. However, there was no statistical difference between adsorption capacity for lysozyme and other high-isoelectric point proteins, suggesting that MIPs are semiselective for this class of proteins. In competitive binding experiments, both MIPs and NIPs preferentially bound lysozyme over other high-isoelectric point proteins. This result demonstrated that imprinting alone could not account for the observed selectivity for lysozyme. Analysis of the solvent accessible surface area of lysozyme and its high-isoelectric point competitors revealed why lysozyme is an exceptional binder to the polymer system used in this work, with or without imprinting.

Charged Poly(N-isopropylacrylamide) Nanogels for the Stabilization of High Isoelectric Point Proteins.

Storage and transportation of protein therapeutics using refrigeration is a costly process; a reliable electrical supply is vital, expensive equipment is needed, and unique transportation is required. Reducing the reliance on the cold chain would enable low-cost transportation and storage of biologics, ultimately improving accessibility of this class of therapeutics to patients in remote locations. Herein, we report on the synthesis of charged poly(N-isopropylacrylamide) nanogels that efficiently adsorb a range of different proteins of varying isoelectric points and molecular weights (e.g., adsorption capacity (Q) = 4.7 ± 0.2 mg/mg at 6 mg/mL initial IgG concentration), provide protection from external environmental factors (i.e., temperature), and subsequently release the proteins in an efficient manner (e.g., 100 ± 1% at 2 mg/mL initial IgG concentration). Both cationic and anionic nanogels were synthesized and selectively chosen based on the ability to form electrostatic interactions with adsorbed proteins (e.g., cationic nanogels adsorb low isoelectric point proteins whereas anionic nanogels adsorb high isoelectric point proteins). The nanogel-protein complex formed upon adsorption increases the stabilization of the protein's tertiary structure, providing protection against denaturation at elevated temperatures (e.g., 84 ± 4% of the protected IgG was stabilized when exposed to 65 °C). The addition of a high molar salt solution (e.g., 40 mM CaCl2 solution) to protein-laden nanogels disrupts the electrostatic interactions and collapses the nanogel, ultimately releasing the protein. The versatile materials utilized, in addition to the protein loading and release mechanisms described, provide a simple and efficient strategy to protect fragile biologics for their transport to remote areas without necessitating costly storage equipment.

Viscoelastic and Thermoreversible Networks Crosslinked by Non-covalent Interactions Between "Clickable" Nucleic Acids Oligomers and DNA.

An approach to efficient and scalable production of oligonucleotide-based gel networks is presented. Specifically, a new class of xenonucleic acid (XNA) synthesized through a scalable and efficient thiol-ene polymerization mechanism, "Clickable" Nucleic Acids (CNAs), were conjugated to a multifunctional poly(ethylene glycol), PEG. In the presence of complementary single stranded DNA (ssDNA), the macromolecular conjugate assembled into a crosslinked 3D gel capable of achieving storage moduli on the order of 1 kPa. Binding studies between the PEG-CNA macromolecule and complementary ssDNA indicate that crosslinking is due to the CNA/DNA interaction. Gel formation was specific to the base sequence and length of the ssDNA crosslinker. The gels were fully thermoreversible, completely melting at temperatures above 60°C and re-forming upon cooling over multiple cycles and with no apparent hysteresis. Shear stress relaxation experiments revealed that relaxation dynamics are dependent on crosslinker length, which is hypothesized to be an effect of the polydisperse CNA chains. Arrhenius analysis of characteristic relaxation times was only possible for shorter crosslinker lengths, and the activation energy for these gels was determined to be 110 ± 20 kJ/mol. Overall, the present work demonstrates that CNA is capable of participating in stimuli-responsive interactions that would be expected from XNAs, and that these interactions support 3D gels that have potential uses in biological and materials science applications.

Efficient cellular uptake of click nucleic acid modified proteins.

Efficient intracellular delivery of biomacromolecules such as proteins continues to remain a challenge despite its potential for medicine. In this work, we show that mScarlet, a non cytotoxic red fluorescent protein (RFP) conjugated to Click Nucleic Acid (CNA), a synthetic analog of DNA, undergo cell uptake significantly more than either native proteins or proteins conjugated with similar amounts of DNA in MDA-MB-468 cells. We further demonstrate that the process of cell uptake is metabolically driven and that scavenger receptors and caveolae mediated endocytosis play a significant role. Co-localization studies using anti-scavenger receptor antibodies suggest that scavenger receptors are implicated in the mechanism of uptake of CNA modified proteins.

Messenger RNA enrichment using synthetic oligo(T) click nucleic acids.

Enrichment of mRNA is a key step in a number of molecular biology techniques, particularly in the rapidly growing field of transcriptomics. Currently, mRNA is isolated using oligo(thymine) DNA (oligo(dT)) immobilized on solid supports, which binds to the poly(A) tail of mRNA to pull the mRNA out of solution through the use of magnets or centrifugal filters. Here, a simple method to isolate mRNA by complexing it with synthetic click nucleic acids (CNAs) is described. Oligo(T) CNA bound efficiently to mRNA, and because of the insolubility of CNA in water, >90% of mRNA was readily removed from solution using this method. Simple washing, buffer exchange, and heating steps enabled mRNA's enrichment from total RNA, with a yield of 3.1 ± 1.5% of the input total RNA by mass, comparable to the yield from commercially available mRNA enrichment beads. Further, the integrity and activity of mRNA after CNA-facilitated pulldown and release was evaluated through two assays. In vitro translation of EGFP mRNA confirmed the translatability of mRNA into functional protein and RT-qPCR was used to amplify enriched mRNA from total RNA extracts and compare gene expression to results obtained using commercially available products.

Label-Free Detection of Tear Biomarkers Using Hydrogel-Coated Gold Nanoshells in a Localized Surface Plasmon Resonance-Based Biosensor.

The dependence of the localized surface plasmon resonance (LSPR) of noble-metal nanomaterials on refractive index makes LSPR a useful, label-free signal transduction strategy for biosensing. In particular, by decorating gold nanomaterials with molecular recognition agents, analytes of interest can be trapped near the surface, resulting in an increased refractive index surrounding the nanomaterial, and, consequently, a red shift in the LSPR wavelength. Ionic poly( N-isopropylacrylamide- co-methacrylic acid) (PNM) hydrogels were used as protein receptors because PNM nanogels exhibit a large increase in refractive index upon protein binding. Specifically, PNM hydrogels were synthesized on the surface of silica gold nanoshells (AuNSs). This composite material (AuNS@PNM) was used to detect changes in the concentration of two protein biomarkers of chronic dry eye: lysozyme and lactoferrin. Both of these proteins have high isoelectric points, resulting in electrostatic attraction between the negatively charged PNM hydrogels and positively charged proteins. Upon binding lysozyme or lactoferrin, AuNS@PNM exhibits large, concentration-dependent red shifts in LSPR wavelength, which enabled the detection of clinically relevant concentration changes of both biomarkers in human tears. The LSPR-based biosensor described herein has potential utility as an affordable screening tool for chronic dry eye and associated conditions.

Protein-Imprinted Polymers: The Shape of Things to Come?

The potential to develop materials with antibody-like molecular recognition properties has helped sustain interest in protein-imprinted polymers over the past several decades. Unfortunately, despite persistent research, the field of noncovalent protein imprinting has seen limited success in terms of achieving materials with high selectivity and high affinity. In this Perspective, important yet sometimes overlooked aspects of the imprinting and binding processes are reviewed to help understand why there has been limited success. In particular, the imprinting and binding processes are viewed through the scope of free radical polymerization and hydrogel swelling theories to underscore the complexity of the synthesis and behavior of protein-imprinted polymers. Additionally, we review the metrics of success commonly used in protein imprinting literature (i.e., adsorption capacity, imprinting factor, and selectivity factor) and consider the relevance of each to the characterization of an imprinted polymer's recognition characteristics. Throughout, common shortcomings are highlighted, and experiments that could help verify or disprove the efficacy of noncovalent protein imprinting are discussed.

Conducting polymer nanoparticles decorated with collagen mimetic peptides for collagen targeting.

We report on the formation of conducting polymer nanoparticles (CPNs), stabilized by a collagen mimetic peptide (CMP)-polymer amphiphile. CPNs ranging from ∼15 to 40 nm were readily accessible upon modifying the amphiphile concentration. Surface presentation of CMPs on CPN precluded intra-/inter-particle trimerization, while preserving their ability to target collagen without pre-activation.