Spatially-Encoding Hydrogels With DNA to Control Cell Signaling.

Patterning biomolecules in synthetic hydrogels offers routes to visualize and learn how spatially-encoded cues modulate cell behavior (e.g., proliferation, differentiation, migration, and apoptosis). However, investigating the role of multiple, spatially defined biochemical cues within a single hydrogel matrix remains challenging because of the limited number of orthogonal bioconjugation reactions available for patterning. Herein, a method to pattern multiple oligonucleotide sequences in hydrogels using thiol-yne photochemistry is introduced. Rapid hydrogel photopatterning of hydrogels with micron resolution DNA features (≈1.5 µm) and control over DNA density are achieved over centimeter-scale areas using mask-free digital photolithography. Sequence-specific DNA interactions are then used to reversibly tether biomolecules to patterned regions, demonstrating chemical control over individual patterned domains. Last, localized cell signaling is shown using patterned protein-DNA conjugates to selectively activate cells on patterned areas. Overall, this work introduces a synthetic method to achieve multiplexed micron resolution patterns of biomolecules onto hydrogel scaffolds, providing a platform to study complex spatially-encoded cellular signaling environments.

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.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo.

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric deformation. Our understanding of the internal relationships between these events has been limited because we can neither 'see' inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue's geometric changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 µm by 540 µm) and the length of a typical trabecula (>2000 µm). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Synthesis and Characterization of Click Nucleic Acid Conjugated Polymeric Microparticles for DNA Delivery Applications.

Microparticle-mediated nucleic acid delivery is a popular strategy to achieve therapeutic outcomes via antisense gene therapy. However, current methods used to fabricate polymeric microparticles suffer from suboptimal properties such as particle polydispersity and low encapsulation efficiency. Here, a new particulate delivery system based on step-growth thiol-Michael dispersion polymerization is reported in which a low polydispersity microparticle is functionalized with a synthetic nucleic acid mimic, namely, click nucleic acids (CNA). CNA oligomers, exhibiting an average length of approximately four nucleic acid repeat units per chain for both adenine and thymine bases, were successfully conjugated to excess thiols present in the microparticles. Effective DNA loading was obtained by simple mixing, and up to 6 ± 2 pmol of complementary DNA/mg of particle was achieved, depending on the length of DNA used. In addition, DNA loading was orders of magnitude less for noncomplementary sequences and sequences containing an alternating base mismatch. The DNA release properties were evaluated, and it was found that release could be triggered by sudden changes in temperature but was unaffected over a range of pH. Finally, phagocytosis of loaded microparticles was observed by confocal microscopy and corroborated by an increase in cellular metabolic activity up to 90%. Overall, this work suggests that CNA functionalized microparticles could be a promising platform for controlled DNA delivery.

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.

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.

Cytocompatibility and Cellular Internalization of PEGylated "Clickable" Nucleic Acid Oligomers.

The recently developed synthetic oligonucleotides referred to as "click" nucleic acids (CNAs) are promising due to their relatively simple synthesis based on thiol-X reactions with numerous potential applications in biotechnology, biodetection, gene silencing, and drug delivery. Here, the cytocompatibility and cellular uptake of rhodamine tagged, PEGylated CNA copolymers (PEG-CNA-RHO) were evaluated. NIH 3T3 fibroblast cells treated for 1 h with 1, 10, or 100 µg/mL PEG-CNA-RHO maintained an average cell viability of 86%, which was not significantly different from the untreated control. Cellular uptake of PEG-CNA-RHO was detected within 30 s, and the amount internalized increased over the course of 1 h. Moreover, these copolymers were internalized within cells to a higher degree than controls consisting of either rhodamine tagged PEG or the rhodamine alone. Uptake was not affected by temperature (i.e., 4 or 37 °C), suggesting a passive uptake mechanism. Subcellular colocalization analysis failed to indicate significant correlations between the internalized PEG-CNA-RHO and the organelles examined (mitochondria, endoplasmic reticulum, endosomes and lysosomes). These results indicate that CNA copolymers are cytocompatible and are readily internalized by cells, supporting the idea that CNAs are a promising alternative to DNA in antisense therapy applications.

Structure and function of a cellulase gene in redclaw crayfish, Cherax quadricarinatus.

The most abundant organic compound produced by plants is cellulose; however, it has long been accepted that most animals do not produce endogenous enzymes required for its degradation, but rely instead on symbiotic relationships with microbes that produce the necessary enzymes. Here, we present the genomic organisation of an endogenous glycosyl hydrolase family (GHF) 9 gene in redclaw crayfish (Cherax quadricarinatus), consolidated from a cDNA sequence determined by Byrne et al. [Gene 239 (1999) 317-324.]. Comparison with several other invertebrate GHF9 genes reveals the conservation of both intron position/phase and splice sequence, which adds support to an argument for an ancestral animal cellulase gene. Furthermore, two introns in plant GHF9 genes are also identical in position, implying a more ancient origin for this class of animal cellulase. Protein purification from redclaw gastric fluid via fast performance liquid chromatography (FPLC) indicated the presence of two endoglucanase enzymes. The molecular weights of these components were determined by matrix-assisted laser desorption/ionisation-time-of-flight (MALDI-TOF) to be 47,887 Da (Cel1) and 50,295 Da (Cel2). Cel1 is possibly the functional product of the described cellulase gene, with N-terminal amino acid residues identical to the translated amino acid sequence from the corresponding gene region. Cel2 was identical to Cel1 for 7 of 11 N-terminal residues and likely to be the product of a paralogous endoglucanase gene. These results suggest that redclaw crayfish possess at least one and possibly two functional, endoglucanase enzymes, although further work is required to confirm their origin and attributes.