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Results for “Bioengineering”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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At least 19 recordsLinked to original sources

bioRxiv2016

Guided self-organization recapitulates tissue architecture in a bioengineered brain organoid model

Recently emerging methodology for generating human tissues in vitro has the potential to revolutionize drug discovery and disease research. Currently, three-dimensional cell culture models either rely on the pronounced ability of mammalian cells to self organize in vitro1-6, or use bioengineered constructs to arrange cells in an organ-like configuration7,8. While self-organizing organoids can recapitulate developmental events at a remarkable level of detail, bioengineered constructs excel at reproducibly generating tissue of a desired architecture. Here, we combine these two approaches to reproducibly generate micropatterned human forebrain tissue while maintaining its self-organizing capacity. We utilize poly(lactide-co-glycolide) copolymer (PLGA) fiber microfilaments as a scaffold to generate elongated embryoid bodies and demonstrate that this influences tissue identity. Micropatterned engineered cerebral organoids (enCORs) display enhanced neuroectoderm formation and improved cortical development. Furthermore, we reconstitute the basement membrane at later stages leading to characteristic cortical tissue architecture including formation of a polarized cortical plate and radial units. enCORs provide the first in vitro system for modelling the distinctive radial organization of the cerebral cortex and allow for the study of neuronal migration. We demonstrate their utility by modelling teratogenic effects of ethanol and show that defects in leading process formation may be responsible for the neuronal migration deficits in fetal alcohol syndrome. Our data demonstrate that combining 3D cell culture with bioengineering can significantly enhance tissue identity and architecture, and establish organoid models for teratogenic compounds.

Developmental Biology↗

bioRxiv2017

Low cost and open source multi-fluorescence imaging system for teaching and research in biology and bioengineering

The advent of easy-to-use open source microcontrollers, off-the-shelf electronics and customizable manufacturing technologies has facilitated the development of inexpensive scientific devices and laboratory equipment. In this study, we describe an imaging system that integrates low-cost and open-source hardware, software and genetic resources. The multi-fluorescence imaging system consists of readily available 470 nm LEDs, a Raspberry Pi camera and a set of filters made with low cost acrylics. This device allows imaging in scales ranging from single colonies to entire plates. We developed a set of genetic components (e.g. promoters, coding sequences, terminators) and vectors following the standard framework of Golden Gate, which allowed the fabrication of genetic constructs in a combinatorial, low cost and robust manner. In order to provide simultaneous imaging of multiple wavelength signals, we screened a series of long stokes shift fluorescent proteins that could be combined with cyan/green fluorescent proteins. We found CyOFP1, mBeRFP and sfGFP to be the most compatible set for 3-channel fluorescent imaging. We developed open source Python code to operate the hardware to run time-lapse experiments with automated control of illumination and camera and a Python module to analyze data and extract meaningful biological information. To demonstrate the potential application of this integral system, we tested its performance on a diverse range of imaging assays often used in disciplines such as microbial ecology, microbiology and synthetic biology. We also assessed its potential for STEM teaching in a high school environment, using it to teach biology, hardware design, optics, and programming. Together, these results demonstrate the successful integration of open source hardware, software, genetic resources and customizable manufacturing to obtain a powerful, low cost and robust system for STEM education, scientific research and bioengineering. All the resources developed here are available under open source licenses.

synthetic biology↗

bioRxiv2016

Bioengineering cobalt chromium cardiovascular stent biomaterial for biofunctionalization

Suboptimal biocompatibility of cardiovascular stents manifest as non-compliance at stent-artery interface in vivo. We optimized a plasma-activated coating (PAC) technology to modify cobalt chromium alloy L605 (PAC-L605) surface of an implantable coronary stent material, for improved biofunctionalization. The PAC-L605 surfaces displayed covalent binding capacity of a protein candidate tropoelastin (TE) by retaining 70.3% of TE after SDS detergent washing. Human coronary artery endothelial cell (HCAEC) proliferation visualized with crystal violet staining, did not vary significantly among the biomaterials at 3 or 5 days. Anchorage of cell cytoskeleton visualized with immunofluorescence and scanning electron microscopy (SEM), showed homogenous cell morphology on PAC/TE (with TE) surfaces. Surface hemocompatibility was assessed with static and flow blood assays, the hydrophilic PAC-L605 displayed lower clot formation compared to L605. Area of surface fibrinogen deposited was significantly lower on PAC-L605 vs. L605. Selected ISO 10993-4 tests for biological evaluation of medical devices in contact with blood indicated significantly lowered plasma markers of thrombin-antithrombin complex (TAT), beta-thromboglobulin ({beta}-TG), soluble P-selectin and soluble terminal complement complex (SC5b-9) on PAC-L605 vs. L605. There was no significant difference for plasma biomarkers of polymorphonuclear elastase (PMN elastase) on PAC-L605 vs. L605. Improved surface biofunctionalization of implantable cardiovascular materials could be achieved by plasma-activated coating (PAC).

Bioengineering↗

bioRxiv2017

Bioengineered intestinal muscularis complexes with long-term spontaneous and periodic contractions

Although critical for studies of gut motility and intestinal regeneration, the in vitro culture of intestinal muscularis with peristaltic function remains a significant challenge. Periodic contractions of intestinal muscularis result from the coordinated activity of smooth muscle cells (SMC), the enteric nervous system (ENS), and interstitial cells of Cajal (ICC). Reproducing this activity requires the preservation of all these cells in one system. Here we report the first serum-free culture methodology that consistently maintains spontaneous and periodic contractions of murine and human intestinal muscularis cells for months. In this system, SMC expressed the mature marker myosin heavy chain, and multipolar/dipolar ICC, uniaxonal/multipolar neurons and glial cells were present. Furthermore, drugs affecting ENS, ICC or SMC altered the contractions. Combining this method with scaffolds, contracting cell sheets were formed with organized architecture. With the addition of intestinal epithelial cells, this platform enabled at least 9 types of cells from mucosa and muscularis to coexist and function. The method constitutes a powerful tool for mechanistic studies of gut motility disorders and the regeneration of full-thickness engineered intestine.\n\nIn the small intestine, the mucosa processes partially digested food and absorbs nutrients while the muscularis actuates the peristaltic flow to transport luminal content aborally. Gut motility is central to its digestive and absorptive function. The intestinal muscularis contains various types of cells: of these, smooth muscle cells, the enteric nervous system (ENS)1,2, and the pacemaker interstitial cells of Cajal (ICC)3 are three important players involved in the development of gut motility. Recent studies on intestinal tissue engineering have highlighted the importance of regenerating the functional intestinal muscularis4-9. A variety of systems derived from different cell sources, including pluripotent stem cells (PSC)4-6, embryonic stem cells (ESC)7 and primary tissue8,9, have been established to accomplish this goal and different contractile activities were developed in these systems. Notably, spontaneous contractions have been generated in culture systems that contained both ICC and smooth muscle cells4,6,10-13. In addition, electrical-induced neurogenic contractions were also successfully produced4,5,8 when ENS was introduced into culture. In one of the most recent studies, both spontaneous contractions and electrical-induced neurogenic contractions were developed in a PSC-based culture system4.

bioengineering↗

bioRxiv2026

Bioengineering of Pea (Pisum sativum) for the Expression of Myoglobin, a Heme-containing Animal Protein

Myoglobin, an oxygen-binding animal protein, was engineered in Pisum sativum (pea) to explore its potential as a food ingredient and balance the amino acid profile. In this study, minimal expression cassettes and binary vectors were used to express bovine myoglobin using particle gun and Agrobacterium-mediated transformation, respectively. Successful integration and expression of the myoglobin gene was achieved in P. sativum, with both methods yielding similar transformation efficiencies (~1%). Expression analysis of T2 seeds revealed that Agrobacterium-mediated transformation-derived transgenic lines that expressed myoglobin under the regulation of a Soybean 7S seed-specific promoter and Tobacco Etch Virus (TEV) translation enhancer and a chimeric Rb7MAR Terminator (Ps-BpRG13 events) consistently yielded the highest level of expression (0.32-1.57% of TSP), while transgenic lines with myoglobin expression under the regulation of a Soybean Phaseolin promoter and Rb7MAR Terminator (Ps-BpRG14 events) resulted in moderate levels of heterologous protein expression (0.13-0.83% TSP). Transgenic events with constitutive 2xCaMV35S promoter, TEV translation enhancer and Rb7MAR terminator (Ps-BpRG15 events) exhibited the lowest level of myoglobin expression (0.09-0.14% TSP). Co-bombardment of two minimal expression cassettes - one with myoglobin under the regulation of the Phaseolin promoter and Rb7MAR Terminator and the other with the nptII selectable marker under the regulation of a 2X constitutive CaMV35S promoter, TEV translational enhancer and TNOS Terminator, yielded lines that exhibited variable expression (0.03-0.77% TSP), with some events comparable in expression to Agrobacterium-derived Ps-pRG14 events. To the best of our knowledge, this is the first report of producing a heme-containing animal protein, myoglobin, in peas, with potential implications for sustainable production of food ingredients and nutritionally fortified and value-added plant products using molecular farming.

plant biology↗

bioRxiv2017

Gene synthesis allows biologists to source genes from farther away in the tree of life

Gene synthesis allows biologists and bioengineers to create novel genetic sequences and codon-optimize transgenes for heterologous expression. Because codon choice is key to gene expression and therefore cellular outcomes, it has been argued that gene synthesis will allow researchers to source genes from organisms that would otherwise have been incompatible, opening up more-distant parts of the tree of life as sources for transgenes. We test if this hypothesis is true for academic biological research using a 10-year data set from Addgene, the non-profit plasmid repository. We observe ~19,000 unique genes deposited to Addgene and classify them by whether they are natural or synthetic using a nucleotide-only technique that we develop here. We find that synthetic genes are an increasing share of Addgene deposits. Most importantly, we find direct evidence that researchers are using gene synthesis to source genes from more genetically-distant organisms, particularly for genes that are longer and thus might otherwise be particularly challenging to express. Thus, we provide the first empirical evidence that gene synthesis is leading biologists and bioengineers to sample more broadly across the rich genetic diversity of life, increasingly making that functionality available for industrial or biomedical advances.

synthetic biology↗

bioRxiv2016

A Pilot Study: Development of a Reference Spatiotemporal Gait Data Set for Indian Subjects.

1.Interpretation of pathological gait has, for decades, offered insight into amputee anomalies and diseases such as cerebral palsy. Gait analysis has been actively used at the Bhagwan Mahaveer Viklang Sahatya Samiti for such purposes. The normative gait data used to compare data obtained from amputees, however, has been collected from laboratories under dissimilar conditions, skewing interpretation. Spatiotemporal gait parameters were extractedfrom 43 male Indian subjects using a 7.5m walkway and the BTS Bioengineering GAITLAB setup. Working under the hypothesis that a lack of cross-cultural validity was somewhat responsible for variations in normative gait, we attempted to develop a region-specific data set for use at the headquarters of the Jaipur Foot Organization Bhagwan Mahaveer Viklang Sahatya Samiti (BMVSS). Stratified random sampling was used to recruit subjects and measures were taken to ensure minimal effects of extraneous variables. Statistical analysis was performed on obtained data using one-way analysis of variance (ANOVA) to gauge the effect age and ethnicity had on normative values of the parameters investigated. We found statistically significant p-values for a few spatiotemporal parameters in the analysis of variance for both age and ethnicity. While the results were much less significant than initially hypothesized, the study proved an efficient way to create a normative gait data set exclusively for use at the gait laboratory of the Jaipur Foot Organization, thereby eliminating potential erroneous interpretation of pathological gait when comparing said gait to normative gait data obtained in laboratories under dissimilar conditions.

bioengineering↗

bioRxiv2017

Refactoring the Genetic Code for Increased Evolvability

The standard genetic code is robust to mutations and base-pairing errors during transcription and translation. Point mutations are most likely to be synonymous or preserve the chemical properties of the original amino acid. Saturation mutagenesis experiments suggest that in some cases the best performing mutant requires a replacement of more than a single nucleotide within a codon. These replacements are essentially inaccessible to common error-based laboratory engineering techniques that alter single nucleotide per mutation event, due to the extreme rarity of adjacent mutations. In this theoretical study, we suggest a radical reordering of the genetic code that maximizes the mutagenic potential of single nucleotide replacements. We explore several possible genetic codes that allow a greater degree of accessibility to the mutational landscape and may result in a hyper-evolvable organism serving as an ideal platform for directed evolution experiments. We then conclude by evaluating potential applications for recoded organisms within the synthetic biology field.\n\nSignificance StatementThe conservative nature of the genetic code prevents bioengineers from efficiently accessing the full mutational landscape of a gene using common error-prone methods. Here we present two computational approaches to generate alternative genetic codes with increased accessibility. These new codes allow mutational transition to a larger pool of amino acids and with a greater degree of chemical differences, using a single nucleotide replacement within the codon, thus increasing evolvability both at the single gene and at the genome levels. Given the widespread use of these techniques for strain and protein improvement along with more fundamental evolutionary biology questions, the use of recoded organisms that maximize evolvability should significantly improve the efficiency of directed evolution, library generation and fitness maximization.

bioengineering↗

bioRxiv2017

Game theoretic consideration of transgenic bacteria in the human gut microbiota as a pro-biotic prophylactic for metabolic syndrome

A game theoretic treatment is introduced to explore the role of nutrition of the gut bacterial microflora as a potential pro-biotic therapy. Rational design of functional foods and nutraceuticals has far reaching public health and therapeutic benefits. Understanding ecological dynamics and how phenotypic manipulations of microbe-microbe interactions of the gut microbiota can provide direct health benefit is currently a fundamental question in bioengineering and more widely in the food, diet and health industries. This work considers a hypothetical adjustment of the microbiome by introducing a transgenic bacterial species that contributes to increased exposure of omega 3 fats in the gut by converting them from the omega 6 fats, dominant in the Western diet. The ratio of the two fats circulating in blood are risk markers, indicators of metabolic syndrome and related conditions. In this work, we consider nutritional exposure to a pro-biotic, a live culture of transgenic bacteria contributing omega 3 fats from omega 6 in the diet. Maintaining a long-term co-existence between native (indigenous) and transgenic bacteria is a challenge. Game theory is the appropriate tool for handling this conflict. The long-term co-existence is guaranteed if the two strains engage in the Snowdrift game. Our game theoretic treatment provides the basis of a model mechanism for prophylactic nutritional therapy for metabolic syndrome by the transgenic bacteria, providing support of indigenous gut microbiota and additional supplementation of a pro-biotic.

bioengineering↗

bioRxiv2018

Probing the Acyl Carrier Protein-Enzyme Interactions within Terminal Alkyne Biosynthetic Machinery

The alkyne functionality has attracted much interest due to its diverse chemical and biological applications. We recently elucidated an acyl carrier protein (ACP)-dependent alkyne biosynthetic pathway, however, little is known about ACP interactions with the alkyne biosynthetic enzymes, an acyl-ACP ligase (JamA) and a membrane-bound bi-functional desaturase/acetylenase (JamB). Here, we showed that JamB has a more stringent interaction with ACP than JamA. In addition, site directed mutagenesis of a non-cognate ACP significantly improved its compatibility with JamB, suggesting a possible electrostatic interaction at the ACP-JamB interface. Finally, error-prone PCR and screening of a second non-cognate ACP identified hot spots on the ACP that are important for interacting with JamB and yielded mutants which were better recognized by JamB. Our data thus not only provide insights into the ACP interactions in alkyne biosynthesis, but it also potentially aids in future combinatorial biosynthesis of alkyne-tagged metabolites for chemical and biological applications.\n\nTopical HeadingBiomolecular Engineering, Bioengineering, Biochemicals, Biofuels, and Food

bioengineering↗

bioRxiv2018

Conversion of iPS derived hepatic progenitors into scalable, functional and developmentally relevant human organoids using an inverted colloidal crystal poly (ethylene glycol) scaffold engineered from collagen-coated pores of defined size

Generation of human organoids from induced pluripotent stem cells (iPSCs) offers exciting possibilities for developmental biology, disease modelling and cell therapy. Significant advances towards those goals have been hampered by dependence on animal derived matrices (e.g. Matrigel), immortalized cell lines and resultant structures that are difficult to control or scale. To address these challenges, we aimed to develop a fully defined liver organoid platform using inverted colloid crystal (ICC) whose 3-dimensional mechanical properties could be engineered to recapitulate the extracellular niche sensed by hepatic progenitors during human development. iPSC derived hepatic progenitors (IH) formed organoids most optimally in ICC scaffolds constructed with 140 {micro}m diameter pores coated with Collagen in a two-step process mimicking liver bud formation. The resultant organoids were closer to adult tissue, compared to 2D and 3D controls, with respect to morphology, gene expression, protein secretion, drug metabolism and viral infection and could integrate, vascularize and function following implantation into livers of immune-deficient mice. Preliminary interrogation of the underpinning mechanisms highlighted the importance of TGF{beta} and hedgehog signalling pathways. The combination of functional relevance with tuneable mechanical properties leads us to propose this bioengineered platform to be ideally suited for a range of future mechanistic and clinical organoid related applications.

bioengineering↗

bioRxiv2018

Compression-Assisted Arthrocentesis of the Knee as a Quality Improvement Intervention

ObjectiveThe present study reports the introduction of mechanical compression of the knee for arthrocentesis as quality improvement intervention in a procedure clinic.\n\nMethods430 consecutive symptomatic osteoarthritic knees underwent arthrocentesis followed by corticosteroid injection (1mg/kg of triamcinolone acetonide). The first 215 consecutive knees underwent conventional arthrocentesis and injection; the quality intervention of a mechanical compression brace was introduced, and the next 215 consecutive knees underwent mechanical compression-assisted arthrocentesis follow by injection. Pain scores, arthrocentesis success, fluid yield, time-to-next-intervention, injections/year, and medical costs were measured.\n\nResultsNo serious adverse events occurred in 430 subjects. Diagnostic synovial fluid ([≥]2 ml) was obtained in 9.3% (20/215) without compression and 40.9% (88/215) with compression (p=0.00001, z for 95% CI= 1.96, Pierson). Mechanical compression was associated with a 231% increase in mean arthrocentesis volume: compression 5.3{+/-}11.2 ml, conventional 1.6{+/-}6.4 ml (CI of difference 2.0 <3.7< 5.4; p=0.00001). Time-to-next-intervention after compression-assisted arthrocentesis was longer: 6.9{+/-}3.5 months compared to conventional: 5.1{+/-}2.7 months (p<0.00001, 95% CI of difference 1.2 <1.8< 2.3). Mechanical compression was associated with a reduction in the number of corticosteroid injections administered per year: mechanical compression: 1.7{+/-}0.9 injections/year; conventional: 2.4{+/-}0.5 injections/year (p<0.00001, 95% CI of difference -0.83 < -0.70< -0.56). Mechanical compression did not increase overall yearly costs associated with management of the symptomatic knee (mechanical compression: $293.30/year/knee, conventional: $373.29/year/knee) (p<0.0001, 95% CI of difference 47 <80< 112).\n\nConclusionsRoutine mechanical compression of the knee for arthrocentesis and injection is an effective bioengineering quality improvement intervention in a procedure clinic.

bioengineering↗

bioRxiv2018

Applicability of Drug Response Metrics for Cancer Studies using Biomaterials

Bioengineers have built increasingly sophisticated models of the tumor microenvironment in which to study cell-cell interactions, mechanisms of cancer growth and metastasis, and to test new potential therapies. These models allow researchers to culture cells in conditions that include features of the in vivo tumor microenvironment (TME) implicated in regulating cancer progression, such as ECM stiffness, integrin binding to the ECM, immune and stromal cells, growth factor and cytokine depots, and a 3D geometry more representative of the TME than tissue culture polystyrene (TCPS). These biomaterials could be particularly useful for drug screening applications to make better predictions of efficacy, offering better translation to preclinical in vivo models and clinical trials. However, it can be challenging to compare drug response reports across different platforms and conditions in the current literature. This is, in part, as a result of inconsistent reporting and use of drug response metrics, and vast differences in cell growth rates across a large variety of biomaterial design. This perspective paper attempts to clarify the definitions of drug response measurements used in the field, and presents examples in which these measurements can and cannot be applied. We suggest as best practice to include appropriate controls, always measure the growth rate of cells in the absence of drug, and follow our provided \"decision tree\" matrix when reporting drug response metrics.

bioengineering↗

bioRxiv2018

An Open-Source Plate Reader

Microplate readers are foundational instruments in experimental biology and bioengineering that enable multiplexed spectrophotometric measurements. To enhance their accessibility, we here report the design, construction, validation, and benchmarking of an open-source microplate reader. The system features full-spectrum absorbance and fluorescence emission detection, in situ optogenetic stimulation, and stand-alone touch screen programming of automated assay protocols. The total system costs <$3500, a fraction of the cost of commercial plate readers, and can detect the fluorescence of common dyes down to [~]10 nanomolar concentration. Functional capabilities were demonstrated in context of synthetic biology, optogenetics, and photosensory biology: by steady-state measurements of ligand-induced reporter gene expression in a model of bacterial quorum sensing, and by flavin photocycling kinetic measurements of a LOV (light-oxygen-voltage) domain photoreceptor used for optogenetic transcriptional activation. Fully detailed guides for assembling the device and automating it using the custom Python-based API (Application Program Interface) are provided. This work contributes a key technology to the growing community-wide infrastructure of open-source biology-focused hardware, whose creation is facilitated by rapid prototyping capabilities and low-cost electronics, optoelectronics, and microcomputers.\n\nTable of Contents Graphic\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC=\"FIGDIR/small/413781_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (30K):\norg.highwire.dtl.DTLVardef@c26eaborg.highwire.dtl.DTLVardef@efb695org.highwire.dtl.DTLVardef@1bc2f41org.highwire.dtl.DTLVardef@1c25c79_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2015

Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies.

Engineered RNAs have become integral components of the synthetic biology and bioengineering toolbox for controlling gene expression. We recently expanded this toolbox by creating small transcription activating RNAs (STARs) that act by disrupting the formation of a target transcriptional terminator hairpin placed upstream of a gene. While STARs are a promising addition to the repertoire of RNA regulators, much work remains to be done to optimize the fold activation of these systems. Here we apply rational RNA engineering strategies to improve the fold activation of two STAR regulators. We demonstrate that a combination of promoter strength tuning and multiple RNA stabilization strategies can improve fold activation from 5.4-fold to 13.4-fold for a STAR regulator derived from the pbuE riboswitch terminator. We then validate the generality of our approach and show that these same strategies improve fold activation from 2.1-fold to 14.6-fold for an unrelated STAR regulator. We also establish that the optimizations preserve the orthogonality of these STARs between themselves and a set of antisense RNA transcriptional repressors, enabling these optimized STARs to be used in more sophisticated circuits. These optimization strategies open the door for creating a generation of additional STARs to use in a broad array of biotechnologies.

Synthetic Biology↗

bioRxiv2016

Network structural features affect stability of Calvin Bassham Benson (CBB) pathway in plants

The stability of the Calvin Bassham Benson (CBB) cycle remains an area of active computational research. Our understanding of biology and the prospect for bioengineered plants with higher productivity may both be impacted by a greater understanding of this area. Here we use the ensemble modelling robustness analysis (EMRA) framework to show that the action of the phosphate/G3P antiporter is much more significant for maintenance of stability than a recently proposed G6P shunt. Additionally, we interpret recent results suggesting that overexpression of RuBiSCO does not improve growth rate of plants but overexpression of SBPase does. Our simulations reproduce this result, but only in models which do not include the G6P shunt. Taken together, these results may suggest a situational role for the G6P shunt, possibly in dynamic situations under starvation or other stress conditions.

Systems Biology↗

bioRxiv2015

Agent-based simulation of large tumors in 3-D microenvironments

Multicellular simulations of tumor growth in complex 3-D tissues, where data come from high content in vitro and bioengineered experiments, have gained significant attention by the cancer modeling community in recent years. Agent-based models are often selected for these problems because they can directly model and track cells states and their interactions with the microenvironment. We describe PhysiCell, a specific agent-based model that includes cell motion, cell cycling, and cell volume changes. The model has been performance tested on systems of 105 cells on desktop computers, and is expected to scale to 106 or more cells on single super-computer compute nodes. We plan an open source release of the software in early 2016 at PhysiCell.MathCancer.org

Cancer Biology↗

bioRxiv2016

Blind tests of RNA nearest neighbor energy prediction

The predictive modeling and design of biologically active RNA molecules requires understanding the energetic balance amongst their basic components. Rapid developments in computer simulation promise increasingly accurate recovery of RNAs nearest neighbor (NN) free energy parameters, but these methods have not been tested in predictive trials or on non-standard nucleotides. Here, we present the first such tests through a RECCES-Rosetta (Reweighting of Energy-function Collection with Conformational Ensemble Sampling in Rosetta) framework that rigorously models conformational entropy, predicts previously unmeasured NN parameters, and estimates these values systematic uncertainties. RECCES-Rosetta recovers the ten NN parameters for Watson-Crick stacked base pairs and thirty-two single-nucleotide dangling-end parameters with unprecedented accuracies - root-mean-square deviations (RMSD) of 0.28 kcal/mol and 0.41 kcal/mol, respectively. For set-aside test sets, RECCES-Rosetta gives an RMSD of 0.32 kcal/mol on eight stacked pairs involving G-U wobble pairs and an RMSD of 0.99 kcal/mol on seven stacked pairs involving non-standard isocytidine-isoguanosine pairs. To more rigorously assess RECCES-Rosetta, we carried out four blind predictions for stacked pairs involving 2,6-diaminopurine-U pairs, which achieved 0.64 kcal/mol RMSD accuracy when tested by subsequent experiments. Overall, these results establish that computational methods can now blindly predict energetics of basic RNA motifs, including chemically modified variants, with consistently better than 1 kcal/mol accuracy. Systematic tests indicate that resolving the remaining discrepancies will require energy function improvements beyond simply reweighting component terms, and we propose further blind trials to test such efforts.\n\nSignificanceUnderstanding RNA machines and how their behavior can be modulated by chemical modification is increasingly recognized as an important biological and bioengineering problem, with continuing discoveries of riboswitches, mRNA regulons, CRISPR-guided editing complexes, and RNA enzymes. Computational strategies to understanding RNA energetics are being proposed, but have not yet faced rigorous tests. We describe a modeling strategy called RECCES-Rosetta that models the full ensemble of motions of RNA in single-stranded form and in helices, including non-standard nucleotides such as 2,6-diaminopurine, a variant of adenosine. When compared to experiments, including blind tests, the energetic accuracies of RECCES-Rosetta calculations are at levels close to experimental error, suggesting that computation can now be used to predict and design basic RNA energetics.

Biophysics↗