Integrative network analysis of miRNA-mRNA expression profiles during epileptogenesis in rats reveals therapeutic targets after emergence of first spontaneous seizure.

Epileptogenesis is the process by which a normal brain becomes hyperexcitable and capable of generating spontaneous recurrent seizures. The extensive dysregulation of gene expression associated with epileptogenesis is shaped, in part, by microRNAs (miRNAs) - short, non-coding RNAs that negatively regulate protein levels. Functional miRNA-mediated regulation can, however, be difficult to elucidate due to the complexity of miRNA-mRNA interactions. Here, we integrated miRNA and mRNA expression profiles sampled over multiple time-points during and after epileptogenesis in rats, and applied bi-clustering and Bayesian modelling to construct temporal miRNA-mRNA-mRNA interaction networks. Network analysis and enrichment of network inference with sequence- and human disease-specific information identified key regulatory miRNAs with the strongest influence on the mRNA landscape, and miRNA-mRNA interactions closely associated with epileptogenesis and subsequent epilepsy. Our findings underscore the complexity of miRNA-mRNA regulation, can be used to prioritise miRNA targets in specific systems, and offer insights into key regulatory processes in epileptogenesis with therapeutic potential for further investigation.

Dual-Targeting of the HER2 Cancer Receptor with an Antibody-Directed Enzyme and a Nanobody-Guided MMAE Prodrug Scaffold.

Antibody-enzyme conjugates have shown potential as tissue-specific prodrug activators by antibody-directed enzyme prodrug therapy (ADEPT), but the approach met challenges clinically due to systemic drug release. Here, we report a novel dual-targeting ADEPT system (DuADEPT) which is based on active cancer receptor targeting of both a trastuzumab-sialidase conjugate (Tz-Sia) and a highly potent sialidase-activated monomethyl auristatin E (MMAE) prodrug scaffold. The scaffold is based on a four-way junction of the artificial nucleic acid analog acyclic (L)-threoninol nucleic acid ((L)-aTNA) which at the ends of its four arms carries one nanobody targeting HER2 and three copies of the prodrug. Dual-targeting of the constructs to two proximal epitopes of HER2 was shown by flow cytometry, and a dual-targeted enzymatic drug release assay revealed cytotoxicity upon prodrug activation specifically for HER2-positive cancer cells. The specific delivery and activation of prodrugs in this way could potentially be used to decrease systemic side effects and increase drug efficacy, and utilization of Tz-Sia provides an opportunity to combine the local chemotherapeutic effect of the DuADEPT with an anticancer immune response.

A functional RNA-origami as direct thrombin inhibitor with fast-acting and specific single-molecule reversal agents in vivo model.

Injectable anticoagulants are widely used in medical procedures to prevent unwanted blood clotting. However, many lack safe, effective reversal agents. Here, we present new data on a previously described RNA origami-based, direct thrombin inhibitor (HEX01). We describe a new, fast-acting, specific, single-molecule reversal agent (antidote) and present in vivo data for the first time, including efficacy, reversibility, preliminary safety, and initial biodistribution studies. HEX01 contains multiple thrombin-binding aptamers appended on an RNA origami. It exhibits excellent anticoagulation activity in vitro and in vivo. The new single-molecule, DNA antidote (HEX02) reverses anticoagulation activity of HEX01 in human plasma within 30 s in vitro and functions effectively in a murine liver laceration model. Biodistribution studies of HEX01 in whole mice using ex vivo imaging show accumulation mainly in the liver over 24 h and with 10-fold lower concentrations in the kidneys. Additionally, we show that the HEX01/HEX02 system is non-cytotoxic to epithelial cell lines and non-hemolytic in vitro. Furthermore, we found no serum cytokine response to HEX01/HEX02 in a murine model. HEX01 and HEX02 represent a safe and effective coagulation control system with a fast-acting, specific reversal agent showing promise for potential drug development.

A Multi-Faceted Binding Assessment of Aptamers Targeting the SARS-CoV-2 Spike Protein.

The COVID-19 pandemic has underscored the critical need for the advancement of diagnostic and therapeutic platforms. These platforms rely on the rapid development of molecular binders that should facilitate surveillance and swift intervention against viral infections. In this study, we have evaluated by three independent research groups the binding characteristics of various published RNA and DNA aptamers targeting the spike protein of the SARS-CoV-2 virus. For this comparative analysis, we have employed different techniques such as biolayer interferometry (BLI), enzyme-linked oligonucleotide assay (ELONA), and flow cytometry. Our data show discrepancies in the reported specificity and affinity among several of the published aptamers and underline the importance of standardized methods, the impact of biophysical techniques, and the controls used for aptamer characterization. We expect our results to contribute to the selection and application of suitable aptamers for the detection of SARS-CoV-2.

The Transcriptional Landscape of Coding and Noncoding RNAs in Recurrent and Nonrecurrent Colon Cancer.

A number of patients with colon cancer with local or local advanced disease suffer from recurrence and there is an urgent need for better prognostic biomarkers in this setting. Here, the transcriptomic landscape of mRNAs, long noncoding RNAs, snRNAs, small nucleolar RNAs (snoRNAs), small Cajal body-specific RNAs, pseudogenes, and circular RNAs, as well as RNAs denoted as miscellaneous RNAs, was profiled by total RNA sequencing. In addition to well-known coding and noncoding RNAs, differential expression analysis also uncovered transcripts that have not been implicated previously in colon cancer, such as RNA5SP149, RNU4-2, and SNORD3A. Moreover, there was a profound global up-regulation of snRNA pseudogenes, snoRNAs, and rRNA pseudogenes in more advanced tumors. A global down-regulation of circular RNAs in tumors relative to normal tissues was observed, although only a few were expressed differentially between tumor stages. Many previously undescribed transcripts, including RNU6-620P, RNU2-20P, VTRNA1-3, and RNA5SP60, indicated strong prognostic biomarker potential in receiver operating characteristics analyses. In summary, this study unveiled numerous differentially expressed RNAs across various classes between recurrent and nonrecurrent colon cancer. Notably, there was a significant global up-regulation of snRNA pseudogenes, snoRNAs, and rRNA pseudogenes in advanced tumors. Many of these newly discovered candidates demonstrate a strong prognostic potential for stage II colon cancer.

Deconvoluting the Effect of Cell-Penetrating Peptides for Enhanced and Controlled Insertion of Large-Scale DNA Nanopores.

DNA nanopores have emerged as powerful tools for molecular sensing, but the efficient insertion of large DNA nanopores into lipid membranes remains challenging. In this study, we investigate the potential of cell-penetrating peptides (CPPs), specifically SynB1 and GALA, to enhance the insertion efficiency of large DNA nanopores. We constructed SynB1- or GALA-functionalized DNA nanopores with an 11 nm inner diameter and visualized and quantified their membrane insertion using a TIRF microscopy-based single-liposome assay. The results demonstrated that incorporating an increasing number of SynB1 or GALA peptides into the DNA nanopore significantly enhanced the membrane perforation. Kinetic analysis revealed that the DNA nanopore scaffold played a role in prearranging the CPPs, which facilitated membrane interaction and pore formation. Notably, the use of pH-responsive GALA peptides allowed highly efficient and pH-controlled insertion of large DNA pores. Furthermore, single-channel recording elucidated that the insertion process of single GALA-modified nanopores into planar lipid bilayers was dynamic, likely forming transient large toroidal pores. Overall, our study highlights the potential of CPPs as insertion enhancers for DNA nanopores, which opens avenues for improved molecule sensing and the controlled release of cargo molecules.

Programmable RNA Loading of Extracellular Vesicles with Toehold-Release Purification.

Synthetic nanoparticles as lipid nanoparticles (LNPs) are widely used as drug delivery vesicles. However, they hold several drawbacks, including low biocompatibility and unfavorable immune responses. Naturally occurring extracellular vesicles (EVs) hold the potential as native, safe, and multifunctional nanovesicle carriers. However, loading of EVs with large biomolecules remains a challenge. Here, we present a controlled loading methodology using DNA-mediated and programmed fusion between EVs and messenger RNA (mRNA)-loaded liposomes. The fusion efficiency is characterized at the single-particle level by real-time microscopy through EV surface immobilization via lipidated biotin-DNA handles. Subsequently, fused EV-liposome particles (EVLs) can be collected by employing a DNA strand-replacement reaction. Transferring the fusion reaction to magnetic beads enables us to scale up the production of EVLs one million times. Finally, we demonstrated encapsulation of mCherry mRNA, transfection, and improved translation using the EVLs compared to liposomes or LNPs in HEK293-H cells. We envision this as an important tool for the EV-mediated delivery of RNA therapeutics.

RNA nanostructures for targeted drug delivery and imaging.

The RNA molecule plays a pivotal role in many biological processes by relaying genetic information, regulating gene expression, and serving as molecular machines and catalyzers. This inherent versatility of RNA has fueled significant advancements in the field of RNA nanotechnology, driving the engineering of complex nanoscale architectures toward biomedical applications, including targeted drug delivery and bioimaging. RNA polymers, serving as building blocks, offer programmability and predictability of Watson-Crick base pairing, as well as non-canonical base pairing, for the construction of nanostructures with high precision and stoichiometry. Leveraging the ease of chemical modifications to protect the RNA from degradation, researchers have developed highly functional and biocompatible RNA architectures and integrated them into preclinical studies for the delivery of payloads and imaging agents. This review offers an educational introduction to the use of RNA as a biopolymer in the design of multifunctional nanostructures applied to targeted delivery in vivo, summarizing physical and biological barriers along with strategies to overcome them. Furthermore, we highlight the most recent progress in the development of both small and larger RNA nanostructures, with a particular focus on imaging reagents and targeted cancer therapeutics in pre-clinical models and provide insights into the prospects of this rapidly evolving field.

ciRS-7 and miR-7 regulate ischemia-induced neuronal death via glutamatergic signaling.

Brain functionality relies on finely tuned regulation of gene expression by networks of non-coding RNAs (ncRNAs) such as the one composed by the circular RNA ciRS-7 (also known as CDR1as), the microRNA miR-7, and the long ncRNA Cyrano. We describe ischemia-induced alterations in the ncRNA network both in vitro and in vivo and in transgenic mice lacking ciRS-7 or miR-7. Our data show that cortical neurons downregulate ciRS-7 and Cyrano and upregulate miR-7 expression during ischemia. Mice lacking ciRS-7 exhibit reduced lesion size and motor impairment, while the absence of miR-7 alone results in increased ischemia-induced neuronal death. Moreover, miR-7 levels in pyramidal excitatory neurons regulate neurite morphology and glutamatergic signaling, suggesting a potential molecular link to the in vivo phenotype. Our data reveal the role of ciRS-7 and miR-7 in modulating ischemic stroke outcome, shedding light on the pathophysiological function of intracellular ncRNA networks in the brain.

Nanopore-Mediated Sequencing of Circular RNA.

Circular RNAs (circRNAs) constitute a group of RNAs defined by a covalent bond between the 5' and 3' end formed by a unique back-splicing event. Most circRNAs are composed of more than one exon, which are spliced together in a linear fashion. This protocol describes methods to sequence full-length circRNA across the back-splicing junction, allowing unambiguous characterization of circRNA-specific exon-intron structures by long-read sequencing (LRS). Two different sequencing approaches are provided: (1) Global circRNA sequencing (the circNick-LRS strategy) relying on circRNA enrichment from total RNA followed by total circRNA long-read sequencing, and (2) targeted circRNA sequencing (the circPanel-LRS strategy) where a preselected panel of circRNA are sequenced without prior circRNA enrichment. Both methods were originally described in Karim et al. (Rahimi et al., Nat Commun 12: 4825, 2021) where they were applied to characterize the exon-intron structure of >10.000 circRNAs in mouse and human brains.

Differential microRNA editing may drive target pathway switching in human temporal lobe epilepsy.

MicroRNAs have emerged as important regulators of the gene expression landscape in temporal lobe epilepsy. The mechanisms that control microRNA levels and influence target choice remain, however, poorly understood. RNA editing is a post-transcriptional mechanism mediated by the adenosine acting on RNA (ADAR) family of proteins that introduces base modification that diversifies the gene expression landscape. RNA editing has been studied for the mRNA landscape but the extent to which microRNA editing occurs in human temporal lobe epilepsy is unknown. Here, we used small RNA-sequencing data to characterize the identity and extent of microRNA editing in human temporal lobe epilepsy brain samples. This detected low-to-high editing in over 40 of the identified microRNAs. Among microRNA exhibiting the highest editing was miR-376a-3p, which was edited in the seed region and this was predicted to significantly change the target pool. The edited form was expressed at lower levels in human temporal lobe epilepsy samples. We modelled the shift in editing levels of miR-376a-3p in human-induced pluripotent stem cell-derived neurons. Reducing levels of the edited form of miR-376a-3p using antisense oligonucleotides resulted in extensive gene expression changes, including upregulation of mitochondrial and metabolism-associated pathways. Together, these results show that differential editing of microRNAs may re-direct targeting and result in altered functions relevant to the pathophysiology of temporal lobe epilepsy and perhaps other disorders of neuronal hyperexcitability.

An Albumin-Holliday Junction Biomolecular Modular Design for Programmable Multifunctionality and Prolonged Circulation.

Combinatorial properties such as long-circulation and site- and cell-specific engagement need to be built into the design of advanced drug delivery systems to maximize drug payload efficacy. This work introduces a four-stranded oligonucleotide Holliday Junction (HJ) motif bearing functional moieties covalently conjugated to recombinant human albumin (rHA) to give a "plug-and-play" rHA-HJ multifunctional biomolecular assembly with extended circulation. Electrophoretic gel-shift assays show successful functionalization and purity of the individual high-performance liquid chromatography-purified modules as well as efficient assembly of the rHA-HJ construct. Inclusion of an epidermal growth factor receptor (EGFR)-targeting nanobody module facilitates specific binding to EGFR-expressing cells resulting in approximately 150-fold increased fluorescence intensity determined by flow cytometric analysis compared to assemblies absent of nanobody inclusion. A cellular recycling assay demonstrated retained albumin-neonatal Fc receptor (FcRn) binding affinity and accompanying FcRn-driven cellular recycling. This translated to a 4-fold circulatory half-life extension (2.2 and 0.55 h, for the rHA-HJ and HJ, respectively) in a double transgenic humanized FcRn/albumin mouse. This work introduces a novel biomolecular albumin-nucleic acid construct with extended circulatory half-life and programmable multifunctionality due to its modular design.

circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA.

Circular RNAs (circRNAs) represent a class of widespread endogenous RNAs that regulate gene expression and thereby influence cell biological decisions with implications for the pathogenesis of several diseases. Here, we disclose a novel gene-regulatory role of circHIPK3 by combining analyses of large genomics datasets and mechanistic cell biological follow-up experiments. Specifically, we use temporal depletion of circHIPK3 or specific RNA binding proteins (RBPs) and identify several perturbed genes by RNA sequencing analyses. Using expression-coupled motif analyses of mRNA expression data from various knockdown experiments, we identify an 11-mer motif within circHIPK3, which is also enriched in genes that become downregulated upon circHIPK3 depletion. By mining eCLIP datasets, we find that the 11-mer motif constitutes a strong binding site for IGF2BP2 and validate this circHIPK3-IGF2BP2 interaction experimentally using RNA-immunoprecipitation and competition assays in bladder cancer cell lines. Our results suggest that circHIPK3 and IGF2BP2 mRNA targets compete for binding. Since the identified 11-mer motif found in circHIPK3 is enriched in upregulated genes following IGF2BP2 knockdown, and since IGF2BP2 depletion conversely globally antagonizes the effect of circHIPK3 knockdown on target genes, our results suggest that circHIPK3 can sequester IGF2BP2 as a competing endogenous RNA (ceRNA), leading to target mRNA stabilization. As an example of a circHIPK3-regulated gene, we focus on the STAT3 mRNA as a specific substrate of IGF2BP2 and validate that manipulation of circHIPK3 regulates IGF2BP2-STAT3 mRNA binding and thereby STAT3 mRNA levels. However, absolute copy number quantifications demonstrate that IGF2BP2 outnumbers circHIPK3 by orders of magnitude, which is inconsistent with a simple 1:1 ceRNA hypothesis. Instead, we show that circHIPK3 can nucleate multiple copies of IGF2BP2, potentially via phase separation, to produce IGF2BP2 condensates. Finally, we show that circHIPK3 expression correlates with overall survival of patients with bladder cancer. Our results are consistent with a model where relatively few cellular circHIPK3 molecules function as inducers of IGF2BP2 condensation thereby regulating STAT3 and other key factors for cell proliferation and potentially cancer progression.

Brain cell-specific origin of circulating microRNA biomarkers in experimental temporal lobe epilepsy.

The diagnosis of epilepsy is complex and challenging and would benefit from the availability of molecular biomarkers, ideally measurable in a biofluid such as blood. Experimental and human epilepsy are associated with altered brain and blood levels of various microRNAs (miRNAs). Evidence is lacking, however, as to whether any of the circulating pool of miRNAs originates from the brain. To explore the link between circulating miRNAs and the pathophysiology of epilepsy, we first sequenced argonaute 2 (Ago2)-bound miRNAs in plasma samples collected from mice subject to status epilepticus induced by intraamygdala microinjection of kainic acid. This identified time-dependent changes in plasma levels of miRNAs with known neuronal and microglial-cell origins. To explore whether the circulating miRNAs had originated from the brain, we generated mice expressing FLAG-Ago2 in neurons or microglia using tamoxifen-inducible Thy1 or Cx3cr1 promoters, respectively. FLAG immunoprecipitates from the plasma of these mice after seizures contained miRNAs, including let-7i-5p and miR-19b-3p. Taken together, these studies confirm that a portion of the circulating pool of miRNAs in experimental epilepsy originates from the brain, increasing support for miRNAs as mechanistic biomarkers of epilepsy.

Spatial Profiling of Circular RNAs in Cancer Reveals High Expression in Muscle and Stromal Cells.

Circular RNAs (circRNA) are covalently closed molecules that can play important roles in cancer development and progression. Hundreds of differentially expressed circRNAs between tumors and adjacent normal tissues have been identified in studies using RNA sequencing or microarrays, emphasizing a strong translational potential. Most previous studies have been performed using RNA from bulk tissues and lack information on the spatial expression patterns of circRNAs. Here, we showed that the majority of differentially expressed circRNAs from bulk tissue analyses of colon tumors relative to adjacent normal tissues were surprisingly not differentially expressed when comparing cancer cells directly with normal epithelial cells. Manipulating the proliferation rates of cells grown in culture revealed that these discrepancies were explained by circRNAs accumulating to high levels in quiescent muscle cells due to their high stability; on the contrary, circRNAs were diluted to low levels in the fast-proliferating cancer cells due to their slow biogenesis rates. Thus, different subcompartments of colon tumors and adjacent normal tissues exhibited striking differences in circRNA expression patterns. Likewise, the high circRNA content in muscle cells was also a strong confounding factor in bulk analyses of circRNAs in bladder and prostate cancers. Together, these findings emphasize the limitations of using bulk tissues for studying differential circRNA expression in cancer and highlight a particular need for spatial analysis in this field of research. SIGNIFICANCE: The abundance of circRNAs varies systematically between subcompartments of solid tumors and adjacent tissues, implying that differentially expressed circRNAs discovered in bulk tissue analyses may reflect differences in cell type composition between samples.

Protein ligand and nanotopography separately drive the phenotype of mouse embryonic stem cells.

Biochemical and biomechanical signals regulate stem cell function in the niche environments in vivo. Current in vitro culture of mouse embryonic stem cells (mESC) uses laminin (LN-511) to provide mimetic biochemical signaling (LN-521 for human systems) to maintain stemness. Alternative approaches propose topographical cues to provide biomechanical cues, however combined biochemical and topographic cues may better mimic the in vivo environment, but are largely unexplored for in vitro stem cell expansion. In this study, we directly compare in vitro signals from LN-511 and/or topographic cues to maintain stemness, using systematically-varied submicron pillar patterns or flat surfaces with or without preadsorbed LN-511. The adhesion of cells, colony formation, expression of the pluripotency marker,octamer-binding transcription factor 4 (Oct4), and transcriptome profiling were characterized. We observed that either biochemical or topographic signals could maintain stemness of mESCs in feeder-free conditions, indicated by high-level Oct4 and gene profiling by RNAseq. The combination of LN-511 with nanotopography reduced colony growth, while maintaining stemness markers, shifted the cellular phenotype indicating that the integration of biochemical and topographic signals is antagonistic. Overall, significantly faster (up to 2.5 times) colony growth was observed at nanotopographies without LN-511, suggesting for improved ESC expansion.

MicroRNA-335-5p suppresses voltage-gated sodium channel expression and may be a target for seizure control.

There remains an urgent need for new therapies for treatment-resistant epilepsy. Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bidirectional control of sodium channel expression are needed. MicroRNAs (miRNA) are small noncoding RNAs which negatively regulate gene expression. Here we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the antiseizure medicine cannabidiol, and plasma from patients with treatment-resistant epilepsy, converge on a single target-miR-335-5p. Pathway analysis on predicted and validated miR-335-5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335-5p resulted in upregulation of Scn1a, Scn2a, and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335-5p. Blocking miR-335-5p also increased voltage-gated sodium currents and SCN1A, SCN2A, and SCN3A expression in human induced pluripotent stem cell-derived neurons. Inhibition of miR-335-5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazol seizure model, whereas adeno-associated virus 9-mediated overexpression of miR-335-5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335-5p may be a means to regulate VGSCs and affect neuronal excitability and seizures. Changes to miR-335-5p may reflect compensatory mechanisms to control excitability and could provide biomarker or therapeutic strategies for different types of treatment-resistant epilepsy.

Specific Detection of Proteins by a Nanobody-Functionalized Nanopore Sensor.

Nanopores are label-free single-molecule analytical tools that show great potential for stochastic sensing of proteins. Here, we described a ClyA nanopore functionalized with different nanobodies through a 5-6 nm DNA linker at its periphery. Ty1, 2Rs15d, 2Rb17c, and nb22 nanobodies were employed to specifically recognize the large protein SARS-CoV-2 Spike, a medium-sized HER2 receptor, and the small protein murine urokinase-type plasminogen activator (muPA), respectively. The pores modified with Ty1, 2Rs15d, and 2Rb17c were capable of stochastic sensing of Spike protein and HER2 receptor, respectively, following a model where unbound nanobodies, facilitated by a DNA linker, move inside the nanopore and provoke reversible blockade events, whereas engagement with the large- and medium-sized proteins outside of the pore leads to a reduced dynamic movement of the nanobodies and an increased current through the open pore. Exploiting the multivalent interaction between trimeric Spike protein and multimerized Ty1 nanobodies enabled the detection of picomolar concentrations of Spike protein. In comparison, detection of the smaller muPA proteins follows a different model where muPA, complexing with the nb22, moves into the pore, generating larger blockage signals. Importantly, the components in blood did not affect the sensing performance of the nanobody-functionalized nanopore, which endows the pore with great potential for clinical detection of protein biomarkers.

Biased activation of the receptor tyrosine kinase HER2.

HER2 belongs to the ErbB sub-family of receptor tyrosine kinases and regulates cellular proliferation and growth. Different from other ErbB receptors, HER2 has no known ligand. Activation occurs through heterodimerization with other ErbB receptors and their cognate ligands. This suggests several possible activation paths of HER2 with ligand-specific, differential response, which has so far remained unexplored. Using single-molecule tracking and the diffusion profile of HER2 as a proxy for activity, we measured the activation strength and temporal profile in live cells. We found that HER2 is strongly activated by EGFR-targeting ligands EGF and TGFα, yet with a distinguishable temporal fingerprint. The HER4-targeting ligands EREG and NRGß1 showed weaker activation of HER2, a preference for EREG, and a delayed response to NRGß1. Our results indicate a selective ligand response of HER2 that may serve as a regulatory element. Our experimental approach is easily transferable to other membrane receptors targeted by multiple ligands.

Parallel Functionalization of DNA Origami.

DNA origami enables the creation of large supramolecular structures, with precisely defined features at the nanoscale. The concept thus naturally lends itself to the concept of molecular patterning, i.e., the positioning of molecular moieties and functional features. Creation of nanoscale patterns was already disseminated by Rothemund in 2006, in which DNA hairpins were used to produce nanoscale patterns on the flat origami canvases (Rothemund PWK, Nature 440(7082):297-302, 2006). For this type of application, it is often desired to produce multiple different patterns using the same origami canvas by reusing existing origami staple strands, rather than ordering new, custom oligonucleotides for each unique pattern. This chapter presents a method where the enzyme terminal deoxynucleotidyl transferase (TdT) is used in a parallelized reaction to add functional moieties to the end of a selected pool of unmodified staple strand oligonucleotides, which are then incorporated at precisely defined positions in the DNA origami canvas. Introducing arrays of functional features using this enzymatic functionalization of origami staple strands offers a very high degree of flexibility, versatility, and ease of use and can often be obtained faster than custom synthesis. For small synthesis scales, typically employed during initial functional screening of many different molecular patterns, the method also offers a significant advantage in terms of cost. During the past years, we have utilized this to incorporate a large variety of molecules including bulky proteins (Sørensen RS, Okholm AH, Schaffert D, Kodal ALB, Gothelf KV, Kjems J, ACS Nano 7:8098-8104, 2013) in designed patterns from modified nucleotide triphosphate (NTP) building blocks (Jahn K, Tørring T, Voigt NV, Sørensen RS, Kodal ALB, Andersen ES, Bioconjug Chem 22:819-823, 2011). The near-quantitative yields obtained by enzymatic functionalization allow synthesis of a large set of oligonucleotides in a one-pot reaction from commercial starting materials without the need for individual post-purification. Based on the chosen subset of staple strand, it is possible to create any designed functionality, array, or pattern. Here we describe the process going from an idea/design of a DNA origami-specific molecular pattern to nucleotide synthesis and subsequent parallel functionalization of the DNA origami, assembly, and the final characterization.