HLA class I signal peptide polymorphism determines the level of CD94/NKG2-HLA-E-mediated regulation of effector cell responses.

Human leukocyte antigen (HLA)-E binds epitopes derived from HLA-A, HLA-B, HLA-C and HLA-G signal peptides (SPs) and serves as a ligand for CD94/NKG2A and CD94/NKG2C receptors expressed on natural killer and T cell subsets. We show that among 16 common classical HLA class I SP variants, only 6 can be efficiently processed to generate epitopes that enable CD94/NKG2 engagement, which we term 'functional SPs'. The single functional HLA-B SP, known as HLA-B/-21M, induced high HLA-E expression, but conferred the lowest receptor recognition. Consequently, HLA-B/-21M SP competes with other SPs for providing epitope to HLA-E and reduces overall recognition of target cells by CD94/NKG2A, calling for reassessment of previous disease models involving HLA-B/-21M. Genetic population data indicate a positive correlation between frequencies of functional SPs in humans and corresponding cytomegalovirus mimics, suggesting a means for viral escape from host responses. The systematic, quantitative approach described herein will facilitate development of prediction algorithms for accurately measuring the impact of CD94/NKG2-HLA-E interactions in disease resistance/susceptibility.

Author Correction: CCR5AS lncRNA variation differentially regulates CCR5, influencing HIV disease outcome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CCR5AS lncRNA variation differentially regulates CCR5, influencing HIV disease outcome.

Multiple genome-wide studies have identified associations between outcome of human immunodeficiency virus (HIV) infection and polymorphisms in and around the gene encoding the HIV co-receptor CCR5, but the functional basis for the strongest of these associations, rs1015164A/G, is unknown. We found that rs1015164 marks variation in an activating transcription factor 1 binding site that controls expression of the antisense long noncoding RNA (lncRNA) CCR5AS. Knockdown or enhancement of CCR5AS expression resulted in a corresponding change in CCR5 expression on CD4+ T cells. CCR5AS interfered with interactions between the RNA-binding protein Raly and the CCR5 3' untranslated region, protecting CCR5 messenger RNA from Raly-mediated degradation. Reduction in CCR5 expression through inhibition of CCR5AS diminished infection of CD4+ T cells with CCR5-tropic HIV in vitro. These data represent a rare determination of the functional importance of a genome-wide disease association where expression of a lncRNA affects HIV infection and disease progression.

Genetic variation that determines TAPBP expression levels associates with the course of malaria in an HLA allotype-dependent manner.

HLA class I (HLA-I) allotypes vary widely in their dependence on tapasin (TAPBP), an integral component of the peptide-loading complex, to present peptides on the cell surface. We identified two single-nucleotide polymorphisms that regulate TAPBP messenger RNA (mRNA) expression in Africans, rs111686073 (G/C) and rs59097151 (A/G), located in an AP-2α transcription factor binding site and a microRNA (miR)-4486 binding site, respectively. rs111686073G and rs59097151A induced significantly higher TAPBP mRNA expression relative to the alternative alleles due to higher affinity for AP-2α and abrogation of miR-4486 binding, respectively. These variants associated with lower Plasmodium falciparum parasite prevalence and lower incidence of clinical malaria specifically among individuals carrying tapasin-dependent HLA-I allotypes, presumably by augmenting peptide loading, whereas tapasin-independent allotypes associated with relative protection, regardless of imputed TAPBP mRNA expression levels. Thus, an attenuated course of malaria may occur through enhanced breadth and/or magnitude of antigen presentation, an important consideration when evaluating vaccine efficacy.

Genome-wide Association Study Identifies HLA-DPB1 as a Significant Risk Factor for Severe Aplastic Anemia.

Severe aplastic anemia (SAA) is a rare disorder characterized by hypoplastic bone marrow and progressive pancytopenia. The etiology of acquired SAA is not understood but is likely related to abnormal immune responses and environmental exposures. We conducted a genome-wide association study of individuals with SAA genetically matched to healthy controls in discovery (359 cases, 1,396 controls) and validation sets (175 cases, 1,059 controls). Combined analyses identified linked SNPs in distinct blocks within the major histocompatibility complex on 6p21. The top SNP encodes p.Met76Val in the P4 binding pocket of the HLA class II gene HLA-DPB1 (rs1042151A>G, odds ratio [OR] 1.75, 95% confidence interval [CI] 1.50-2.03, p = 1.94 × 10-13) and was associated with HLA-DP cell surface expression in healthy individuals (p = 2.04 × 10-6). Phylogenetic analyses indicate that Val76 is not monophyletic and likely occurs in conjunction with different HLA-DP binding groove conformations. Imputation of HLA-DPB1 alleles revealed increased risk of SAA associated with Val76-encoding alleles DPB1∗03:01, (OR 1.66, p = 1.52 × 10-7), DPB1∗10:01 (OR 2.12, p = 0.0003), and DPB1∗01:01 (OR 1.60, p = 0.0008). A second SNP near HLA-B, rs28367832G>A, reached genome-wide significance (OR 1.49, 95% CI 1.22-1.78, p = 7.27 × 10-9) in combined analyses; the association remained significant after excluding cases with clonal copy-neutral loss-of-heterozygosity affecting class I HLA genes (8.6% of cases and 0% of controls). SNPs in the HLA class II gene HLA-DPB1 and possibly class I (HLA-B) are associated with SAA. The replacement of Met76 to Val76 in certain HLA-DPB1 alleles might influence risk of SAA through mechanisms involving DP peptide binding specificity, expression, and/or other factors affecting DP function.

HLA tapasin independence: broader peptide repertoire and HIV control.

Human leukocyte antigen (HLA) class I allotypes vary in their ability to present peptides in the absence of tapasin, an essential component of the peptide loading complex. We quantified tapasin dependence of all allotypes that are common in European and African Americans (n = 97), which revealed a broad continuum of values. Ex vivo examination of cytotoxic T cell responses to the entire HIV-1 proteome from infected subjects indicates that tapasin-dependent allotypes present a more limited set of distinct peptides than do tapasin-independent allotypes, data supported by computational predictions. This suggests that variation in tapasin dependence may impact the strength of the immune responses by altering peptide repertoire size. In support of this model, we observed that individuals carrying HLA class I genotypes characterized by greater tapasin independence progress more slowly to AIDS and maintain lower viral loads, presumably due to increased breadth of peptide presentation. Thus, tapasin dependence level, like HLA zygosity, may serve as a means to restrict or expand breadth of the HLA-I peptide repertoire across humans, ultimately influencing immune responses to pathogens and vaccines.

HLA-A*03 and response to immune checkpoint blockade in cancer: an epidemiological biomarker study.

BACKGROUND: Predictive biomarkers could allow more precise use of immune checkpoint inhibitors (ICIs) in treating advanced cancers. Given the central role of HLA molecules in immunity, variation at the HLA loci could differentially affect the response to ICIs. The aim of this epidemiological study was to determine the effect of HLA-A*03 as a biomarker for predicting response to immunotherapy. METHODS: In this epidemiological study, we investigated the clinical outcomes (overall survival, progression free survival, and objective response rate) after treatment for advanced cancer in eight cohorts of patients: three observational cohorts of patients with various types of advanced tumours (the Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets [MSK-IMPACT] cohort, the Dana-Farber Cancer Institute [DFCI] Profile cohort, and The Cancer Genome Atlas) and five clinical trials of patients with advanced bladder cancer (JAVELIN Solid Tumour) or renal cell carcinoma (CheckMate-009, CheckMate-010, CheckMate-025, and JAVELIN Renal 101). In total, these cohorts included 3335 patients treated with various ICI agents (anti-PD-1, anti-PD-L1, and anti-CTLA-4 inhibitors) and 10 917 patients treated with non-ICI cancer-directed therapeutic approaches. We initially modelled the association of HLA amino-acid variation with overall survival in the MSK-IMPACT discovery cohort, followed by a detailed analysis of the association between HLA-A*03 and clinical outcomes in MSK-IMPACT, with replication in the additional cohorts (two further observational cohorts and five clinical trials). FINDINGS: HLA-A*03 was associated in an additive manner with reduced overall survival after ICI treatment in the MSK-IMPACT cohort (HR 1·48 per HLA-A*03 allele [95% CI 1·20-1·82], p=0·00022), the validation DFCI Profile cohort (HR 1·22 per HLA-A*03 allele, 1·05-1·42; p=0·0097), and in the JAVELIN Solid Tumour clinical trial for bladder cancer (HR 1·36 per HLA-A*03 allele, 1·01-1·85; p=0·047). The HLA-A*03 effect was observed across ICI agents and tumour types, but not in patients treated with alternative therapies. Patients with HLA-A*03 had shorter progression-free survival in the pooled patient population from the three CheckMate clinical trials of nivolumab for renal cell carcinoma (HR 1·31, 1·01-1·71; p=0·044), but not in those receiving control (everolimus) therapies. Objective responses were observed in none of eight HLA-A*03 homozygotes in the ICI group (compared with 59 [26·6%] of 222 HLA-A*03 non-carriers and 13 (17·1%) of 76 HLA-A*03 heterozygotes). HLA-A*03 was associated with shorter progression-free survival in patients receiving ICI in the JAVELIN Renal 101 randomised clinical trial for renal cell carcinoma (avelumab plus axitinib; HR 1·59 per HLA-A*03 allele, 1·16-2·16; p=0·0036), but not in those receiving control (sunitinib) therapy. Objective responses were recorded in one (12·5%) of eight HLA-A*03 homozygotes in the ICI group (compared with 162 [63·8%] of 254 HLA-A*03 non-carriers and 40 [55·6%] of 72 HLA-A*03 heterozygotes). HLA-A*03 was associated with impaired outcome in meta-analysis of all 3335 patients treated with ICI at genome-wide significance (p=2·01 × 10-8) with no evidence of heterogeneity in effect (I2 0%, 95% CI 0-0·76) INTERPRETATION: HLA-A*03 is a predictive biomarker of poor response to ICI. Further evaluation of HLA-A*03 is warranted in randomised trials. HLA-A*03 carriage could be considered in decisions to initiate ICI in patients with cancer. FUNDING: National Institutes of Health, Merck KGaA, and Pfizer.

Association of Inhibitory Killer Cell Immunoglobulin-like Receptor Ligands With Higher Plasmodium falciparum Parasite Prevalence.

Killer cell immunoglobulin-like receptors (KIRs) and their HLA ligands influence the outcome of many infectious diseases. We analyzed the relationship of compound KIR-HLA genotypes with risk of Plasmodium falciparum infection in a longitudinal cohort of 890 Ugandan individuals. We found that presence of HLA-C2 and HLA-Bw4, ligands for inhibitory KIR2DL1 and KIR3DL1, respectively, increased the likelihood of P. falciparum parasitemia in an additive manner. Individuals homozygous for HLA-C2, which mediates strong inhibition via KIR2DL1, had the highest odds of parasitemia, HLA-C1/C2 heterozygotes had intermediate odds, and individuals homozygous for HLA-C1, which mediates weaker inhibition through KIR2DL2/3, had the lowest odds of parasitemia. In addition, higher surface expression of HLA-C, the ligand for inhibitory KIR2DL1/2/3, was associated with a higher likelihood of parasitemia. Together these data indicate that stronger KIR-mediated inhibition confers a higher risk of P. falciparum parasitemia and suggest that KIR-expressing effector cells play a role in mediating antiparasite immunity.

HLA Zygosity Increases Risk of Hepatitis B Virus-Associated Hepatocellular Carcinoma.

BACKGROUND: Diversity in the HLA genes might be associated with disease outcomes-the heterozygote advantage hypothesis. We tested this hypothesis in relation to hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC). METHODS: We utilized DNA from > 10 000 Taiwanese individuals with current or past HBV infection to examine the association between HLA diversity and critical natural history steps in the progression from HBV infection to HCC. Individuals were classified as homozygotes at a given locus when imputed to carry the same 4-digit allele for the 2 HLA alleles at that locus. RESULTS: Increase in number of homozygous HLA class II loci was associated with an increased risk of chronic HBV infection (Ptrend = 1.18 × 10-7). Among chronic HBV carriers, increase in number of homozygous HLA class II loci was also associated with an increased risk of HBV-associated HCC (Ptrend = .031). For individual HLA loci, HLA-DQB1 homozygosity was significantly associated with HCC risk (adjusted hazard ratio = 1.40; 95% confidence interval, 1.06-1.84). We also found that zygosity affects risk of HCC through its ability to affect viral control. CONCLUSIONS: Homozygosity at HLA class II loci, particularly HLA-DQB1, is associated with a higher risk of HBV-associated HCC.

RNA-DNA fibers and polygons with controlled immunorecognition activate RNAi, FRET and transcriptional regulation of NF-κB in human cells.

Nucleic acid-based assemblies that interact with each other and further communicate with the cellular machinery in a controlled manner represent a new class of reconfigurable materials that can overcome limitations of traditional biochemical approaches and improve the potential therapeutic utility of nucleic acids. This notion enables the development of novel biocompatible 'smart' devices and biosensors with precisely controlled physicochemical and biological properties. We extend this novel concept by designing RNA-DNA fibers and polygons that are able to cooperate in different human cell lines and that have defined immunostimulatory properties confirmed by ex vivo experiments. The mutual intracellular interaction of constructs results in the release of a large number of different siRNAs while giving a fluorescent response and activating NF-κB decoy DNA oligonucleotides. This work expands the possibilities of nucleic acid technologies by (i) introducing very simple design principles and assembly protocols; (ii) potentially allowing for a simultaneous release of various siRNAs together with functional DNA sequences and (iii) providing controlled rates of reassociation, stabilities in human blood serum, and immunorecognition.

Photoactivation of sulfonated polyplexes enables localized gene silencing by DsiRNA in breast cancer cells.

Translation potential of RNA interference nanotherapeutics remains challenging due to in vivo off-target effects and poor endosomal escape. Here, we developed novel polyplexes for controlled intracellular delivery of dicer substrate siRNA, using a light activation approach. Sulfonated polyethylenimines covalently linked to pyropheophorbide-α for photoactivation and bearing modified amines (sulfo-pyro-PEI) for regulated endosomal escape were investigated. Gene knock-down by the polymer-complexed DsiRNA duplexes (siRNA-NPs) was monitored in breast cancer cells. Surprisingly, sulfo-pyro-PEI/siRNA-NPs failed to downregulate the PLK1 or eGFP proteins. However, photoactivation of these cell associated-polyplexes with a 661-nm laser clearly restored knock-down of both proteins. In contrast, protein down-regulation by non-sulfonated pyro-PEI/siRNA-NPs occurred without any laser treatments, indicating cytoplasmic disposition of DsiRNA followed a common intracellular release mechanism. Therefore, sulfonated pyro-PEI holds potential as a unique trap and release light-controlled delivery platform for on-demand gene silencing bearing minimal off target effects.

A cationic amphiphilic co-polymer as a carrier of nucleic acid nanoparticles (Nanps) for controlled gene silencing, immunostimulation, and biodistribution.

Programmable nucleic acid nanoparticles (NANPs) provide controlled coordination of therapeutic nucleic acids (TNAs) and other biological functionalities. Beyond multivalence, recent reports demonstrate that NANP technology can also elicit a specific immune response, adding another layer of customizability to this innovative approach. While the delivery of nucleic acids remains a challenge, new carriers are introduced and tested continuously. Polymeric platforms have proven to be efficient in shielding nucleic acid cargos from nuclease degradation while promoting their delivery and intracellular release. Here, we venture beyond the delivery of conventional TNAs and combine the stable cationic poly-(lactide-co-glycolide)-graft-polyethylenimine with functionalized NANPs. Furthermore, we compare several representative NANPs to assess how their overall structures influence their delivery with the same carrier. An extensive study of various formulations both in vitro and in vivo reveals differences in their immunostimulatory activity, gene silencing efficiency, and biodistribution, with fibrous NANPs advancing for TNA delivery.

Pre-clinical compartmental pharmacokinetic modeling of 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) as a photosensitizer in rat plasma by validated HPLC method.

A second-generation chlorin-based photosensitizer, 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) has shown tremendous therapeutic potential in clinical trials in the treatment of esophageal cancer. Herein, we have developed and validated a bioanalytical method for estimation of HPPH in rat plasma using High Performance Liquid Chromatography (HPLC) with a photo diode array (PDA) detector. The method was applied for carrying out pharmacokinetic study of HPPH. Further pharmacokinetic modeling was carried out to understand the compartment kinetics of HPPH. The developed method was fully validated as per the United States Food and Drug Administration (US-FDA) guidelines for bioanalytical method validation. The linearity of the method was in the range of 250-8000 ng mL-1, and the plasma recovery was found to be 70%. Pharmacokinetic parameters were evaluated and compared via non-compartment analysis and compartment modeling after the intravenous (i.v.) bolus administration in rats using Phoenix WinNonlin 8.0 (Certara™, USA). From the obtained results, we hypothesize that the HPPH complies with two compartmental pharmacokinetic model. Furthermore, it was observed that HPPH has the rapid distribution from the central compartment to peripheral compartment along with slow elimination from peripheral compartment.

Functionally-interdependent shape-switching nanoparticles with controllable properties.

We introduce a new concept that utilizes cognate nucleic acid nanoparticles which are fully complementary and functionally-interdependent to each other. In the described approach, the physical interaction between sets of designed nanoparticles initiates a rapid isothermal shape change which triggers the activation of multiple functionalities and biological pathways including transcription, energy transfer, functional aptamers and RNA interference. The individual nanoparticles are not active and have controllable kinetics of re-association and fine-tunable chemical and thermodynamic stabilities. Computational algorithms were developed to accurately predict melting temperatures of nanoparticles of various compositions and trace the process of their re-association in silico. Additionally, tunable immunostimulatory properties of described nanoparticles suggest that the particles that do not induce pro-inflammatory cytokines and high levels of interferons can be used as scaffolds to carry therapeutic oligonucleotides, while particles with strong interferon and mild pro-inflammatory cytokine induction may qualify as vaccine adjuvants. The presented concept provides a simple, cost-effective and straightforward model for the development of combinatorial regulation of biological processes in nucleic acid nanotechnology.

RNA Fibers as Optimized Nanoscaffolds for siRNA Coordination and Reduced Immunological Recognition.

RNA is a versatile biomaterial that can be used to engineer nanoassemblies for personalized treatment of various diseases. Despite promising advancements, the design of RNA nanoassemblies with minimal recognition by the immune system remains a major challenge. Here, an approach is reported to engineer RNA fibrous structures to operate as a customizable platform for efficient coordination of siRNAs and for maintaining low immunostimulation. Functional RNA fibers are studied in silico and their formation is confirmed by various experimental techniques and visualized by atomic force microscopy (AFM). It is demonstrated that the RNA fibers offer multiple advantages among which are: i) programmability and modular design that allow for simultaneous controlled delivery of multiple siRNAs and fluorophores, ii) reduced immunostimulation when compared to other programmable RNA nanoassemblies, and iii) simple production protocol for endotoxin-free fibers with the option of their cotranscriptional assembly. Furthermore, it is shown that functional RNA fibers can be efficiently delivered with various organic and inorganic carriers while retaining their structural integrity in cells. Specific gene silencing triggered by RNA fibers is assessed in human breast cancer and melanoma cell lines, with the confirmed ability of functional fibers to selectively target single nucleotide mutations.

Dynamic Behavior of RNA Nanoparticles Analyzed by AFM on a Mica/Air Interface.

RNA is an attractive biopolymer for engineering self-assembling materials suitable for biomedical applications. Previously, programmable hexameric RNA rings were developed for the controlled delivery of up to six different functionalities. To increase the potential for functionalization with little impact on nanoparticle topology, we introduce gaps into the double-stranded regions of the RNA rings. Molecular dynamic simulations are used to assess the dynamic behavior and the changes in the flexibility of novel designs. The changes suggested by simulations, however, cannot be clearly confirmed by the conventional techniques such as nondenaturing polyacrylamide gel electrophoresis (native-PAGE) and dynamic light scattering (DLS). Also, an in vitro analysis in primary cultures of human peripheral blood mononuclear cells does not reveal any discrepancy in the immunological recognition of new assemblies. To address these deficiencies, we introduce a computer-assisted quantification strategy. This strategy is based on an algorithmic atomic force microscopy (AFM)-resolved deformation analysis of the RNA nanoparticles studied on a mica/air interface. We validate this computational method by manual image analysis and fitting it to the simulation-predicted results. The presented nanoparticle modification strategy and subsequent AFM-based analysis are anticipated to provide a broad spectrum approach for the future development of nucleic acid-based nanotechnology.

Design and biological activity of novel stealth polymeric lipid nanoparticles for enhanced delivery of hydrophobic photodynamic therapy drugs.

Advances in in vivo stability and preferential tumor uptake of cancer nanomedicine are warranted for effective chemotherapy. Here, we describe a novel nanoformulation using an unconventional polymeric tubule-forming phospholipid, DC8,9PC. We report that DC8,9PC transitions to stable vesicles (LNPs) in the presence of PEGylated lipid (DSPE-PEG2000); the resulting DC8,9PC:DSPE-PEG2000 LNPs efficiently included a hydrophobic PDT drug, HPPH. Remarkably, these LNPs incorporated unusually high DSPE-PEG2000 concentrations; LNP10-HPPH and LNP20-HPPH (10 & 20 mol% PEGylated lipid, respectively) exhibited >90% serum stability at 37 °C. Increased PEGylation in the LNPs correlated with enhanced tumor accumulation in intravenously injected HT29 tumor mouse xenographs. Colon-26 bearing BALB/c mice, intravenously injected with LNP20-HPPH showed superior PDT efficacy and animal survival (no tumor recurrence up to 100 days) as compared to a formulation currently used in clinical trials. Taken together, we present a simple stealth binary lipid nanosystem with enhanced efficiency of tumor accumulation and superior therapeutic efficacy.

Versatile RNA tetra-U helix linking motif as a toolkit for nucleic acid nanotechnology.

RNA nanotechnology employs synthetically modified ribonucleic acid (RNA) to engineer highly stable nanostructures in one, two, and three dimensions for medical applications. Despite the tremendous advantages in RNA nanotechnology, unmodified RNA itself is fragile and prone to enzymatic degradation. In contrast to use traditionally modified RNA strands e.g. 2'-fluorine, 2'-amine, 2'-methyl, we studied the effect of RNA/DNA hybrid approach utilizing a computer-assisted RNA tetra-uracil (tetra-U) motif as a toolkit to address questions related to assembly efficiency, versatility, stability, and the production costs of hybrid RNA/DNA nanoparticles. The tetra-U RNA motif was implemented to construct four functional triangles using RNA, DNA and RNA/DNA mixtures, resulting in fine-tunable enzymatic and thermodynamic stabilities, immunostimulatory activity and RNAi capability. Moreover, the tetra-U toolkit has great potential in the fabrication of rectangular, pentagonal, and hexagonal NPs, representing the power of simplicity of RNA/DNA approach for RNA nanotechnology and nanomedicine community.

Multistrand Structure Prediction of Nucleic Acid Assemblies and Design of RNA Switches.

RNA is an attractive material for the creation of molecular logic gates that release programmed functionalities only in the presence of specific molecular interaction partners. Here we present HyperFold, a multistrand RNA/DNA structure prediction approach for predicting nucleic acid complexes that can contain pseudoknots. We show that HyperFold also performs competitively compared to other published folding algorithms. We performed a large variety of RNA/DNA hybrid reassociation experiments for different concentrations, DNA toehold lengths, and G+C content and find that the observed tendencies for reassociation correspond well to computational predictions. Importantly, we apply this method to the design and experimental verification of a two-stranded RNA molecular switch that upon binding to a single-stranded RNA toehold disease-marker trigger mRNA changes its conformation releasing an shRNA-like Dicer substrate structure. To demonstrate the concept, connective tissue growth factor (CTGF) mRNA and enhanced green fluorescent protein (eGFP) mRNA were chosen as trigger and target sequences, respectively. In vitro experiments confirm the formation of an RNA switch and demonstrate that the functional unit is being released when the trigger RNA interacts with the switch toehold. The designed RNA switch is shown to be functional in MDA-MB-231 breast cancer cells. Several other switches were also designed and tested. We conclude that this approach has considerable potential because, in principle, it allows the release of an siRNA designed against a gene that differs from the gene that is utilized as a biomarker for a disease state.

The Use of Minimal RNA Toeholds to Trigger the Activation of Multiple Functionalities.

Current work reports the use of single-stranded RNA toeholds of different lengths to promote the reassociation of various RNA-DNA hybrids, which results in activation of multiple split functionalities inside human cells. The process of reassociation is analyzed and followed with a novel computational multistrand secondary structure prediction algorithm and various experiments. All of our previously designed RNA/DNA nanoparticles employed single-stranded DNA toeholds to initiate reassociation. The use of RNA toeholds is advantageous because of the simpler design rules, the shorter toeholds, and the smaller size of the resulting nanoparticles (by up to 120 nucleotides per particle) compared to the same hybrid nanoparticles with single-stranded DNA toeholds. Moreover, the cotranscriptional assemblies result in higher yields for hybrid nanoparticles with ssRNA toeholds.