About

Aaron Engelhart, PhD, is an Associate Professor affiliated with the Department of Genetics, Cell Biology & Development at the University of Minnesota Medical School. His professional role involves research and teaching within these fields, contributing to the academic community through his expertise. Specific details about his research focus, background, or key contributions are not provided in the available page text.

Research topics

• Computer Science
• Biology
• Genetics
• Bioinformatics
• Astronomy
• Geography
• Pathology
• Risk analysis (engineering)
• Evolutionary biology
• Astrobiology
• Medicine
• Intensive care medicine
• Data science
• Virology
• Computational biology
• Archaeology
• Cell biology

Selected publications

• Purification of post-transcriptionally modified tRNAs for enhanced cell-free translation systems

  Nucleic Acids Research · 2026-02-24 · 1 citations

  articleOpen access

  Transfer RNAs (tRNAs) are utilized by the ribosome to decode the nucleic acid alphabet. tRNA structure, stability, aminoacylation efficiency, and decoding efficacy are governed by their extensive post-transcriptional modifications. In most studies, individual tRNAs are generated using in vitro transcription, which produces tRNAs devoid of these critical site-specific modifications, negatively affecting translation yields and fidelity. To address this challenge, we have developed a purification method that couples tRNA overexpression to DNA hybridization-based purification. Using this approach, we produced native tRNAs from Escherichia coli in high yield and purity while retaining their complement of native post-transcriptional modifications and translational activity. We extend this technique to the purification of Mj-$tRNA_{CUA}^{Opt}$ and Ma-$tRNA_{CUA}^{Pyl}$, tRNAs of critical importance for genetic code expansion. We confirmed that both Mj-$tRNA_{CUA}^{Opt}$ and Ma-$tRNA_{CUA}^{Pyl}$ contain native E. coli post-transcriptional modifications and provide the first complete modification profiles of each. Moreover, we found that in vivo-generated Mj-$tRNA_{CUA}^{Opt}$ and Ma-$tRNA_{CUA}^{Pyl}\ $significantly outperform their in vitro-generated counterparts in amber codon suppression in cell-free translation reactions. Finally, we purified an engineered variant of E. coli$tRNA_{CCA}^{Trp}$, extending our studies to synthetic tRNAs. We present a flexible method that generates modified tRNAs in high yield and purity, addressing a critical and persistent challenge in RNA biochemistry.

  PublisherDOI

• High Yield, Low Magnesium Flexizyme Reactions in a Water-Ice Eutectic Phase

  Biochemistry · 2025-08-20 · 1 citations

  article

  Flexizymes enable the stoichiometric acylation of tRNAs with a variety of compounds, enabling the in vitro translation of peptides with both non-natural backbones and side chains. However, flexizyme reactions have several drawbacks, including single-turnover kinetics, high Mg(II) carryover, inhibiting in vitro translation, and rapid product hydrolysis. Here we present flexizyme reactions utilizing an ice-eutectic phase, with high yields, 30 times lower Mg(II), and long-term product stability. The eutectic flexizyme reactions increase the ease of use, yield and flexibility of aminoacylation and significantly increase the in vitro protein production.

  PublisherDOI

• Purification of post-transcriptionally modified tRNAs for enhanced cell-free translation systems

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-06-10 · 1 citations

  preprintOpen access

  Abstract Transfer RNAs (tRNAs) are utilized by the ribosome to decode the nucleic acid alphabet. tRNA structure, stability, aminoacylation efficiency, and decoding efficacy are governed by their extensive post-transcriptional modifications. In most studies, individual tRNAs are generated using in vitro transcription, which produces tRNAs devoid of these critical site-specific modifications, negatively affecting translation yields and fidelity. To address this, we have developed a purification method which couples tRNA overexpression to DNA hybridization-based purification. Using this approach, we produced native tRNAs from E. coli in high yield and purity while retaining their complement of native post-transcriptional modifications and translational activity. We extend this technique to the purification of and , tRNAs of critical importance for genetic code expansion. We confirmed that both and contain native E. coli post-transcriptional modifications and provide the first complete modification profiles of each. Moreover, we found that in vivo- generated significantly outperforms its in vitro- generated counterpart in amber codon suppression in cell-free translation reactions. Finally, we purified an engineered variant of E. coli , extending our studies to synthetic tRNAs. We present a flexible method which generates modified tRNAs in high yield and purity, addressing a critical and persistent challenge in RNA biochemistry. This toolkit enables future structural and cell-free studies through scalable access to native and engineered tRNAs, advancing the broader field of translation and synthetic biology.

  PublisherDOI

• One-pot cloning and protein expression platform for genetic engineering

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-08-29 · 1 citations

  preprintOpen access

  In this work, we present a streamlined one-pot cloning and protein expression platform that integrates mutagenesis, plasmid assembly, and functional protein testing in a single reaction. By combining Golden Gate cloning with cell-free transcription-translation, we demonstrate efficient generation and screening of genetic variants without the need for intermediate purification or bacterial amplification. Using fluorescent proteins, luciferase enzymes, antibiotic-converting enzymes, and the violacein biosynthetic pathway, we validate the versatility of this approach for single- and multi-site mutagenesis, combinatorial variant libraries, metabolic pathway programming, and whole-plasmid assembly. By demonstrating compatibility with multiplexed reactions and multi-cistronic constructs, we establish this approach as a generalizable and automatable method for high-throughput cloning and protein engineering in synthetic biology.

  PublisherOA PDFDOI

• An Expanded Repertoire of tRNA Sources for Cell-Free Protein Synthesis

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-08-21 · 1 citations

  preprintOpen access

  Abstract Cell-free expression systems (CFE) are flexible protein translation platforms that simplify the central dogma into an accessible reaction space. Within these systems, bulk transfer RNAs (tRNAs) are critical substrates which deliver amino acids to the elongating ribosome. For years, CFE systems were completed with commercially available tRNA isolated from E. coli MRE600. All commercial sources of tRNA have since been discontinued, jeopardizing future work in all applications of cell-free translation. Here, we address this need by repurposing previously described tRNA isolation methods to produce tRNAs suitable for CFE applications. We isolated the tRNA pools of E. coli strains A19, BL21(DE3), and Rosetta2 BL21(DE3), finding A19 tRNAs but not BL21(DE3) or Rosetta2 BL21(DE3) capable of robust in vitro translation. We determined the abundances of individual tRNAs using tRNA-seq, finding BL21(DE3) and Rosetta2 BL21(DE3) contained outsized abundances of several tRNAs, compromising translation activity. Using codon optimization strategies which align codon usage to tRNA abundance, we were able to mitigate the impact of misaligned tRNA abundances. We extended these studies to V. natriegens , a promising platform for synthetic biology and CFE. We find that neither exogenous V. natriegens tRNAs nor codon optimization are viable options to improve translation yields. Our work here highlights the importance of tRNA abundance within the context of CFE, and simultaneously addresses a critical challenge within cell-free translation.

  PublisherOA PDFDOI

• Quencher-Free Fluorescence Monitoring of G-Quadruplex Folding

  ACS Omega · 2025-01-15 · 3 citations

  articleOpen accessSenior authorCorresponding

  Guanine-rich sequences exhibit a high degree of polymorphism and can form single-stranded, Watson-Crick duplex, and four-stranded G-quadruplex structures. These sequences have found a wide range of uses in synthetic biology applications, arising in part from their structural plasticity. High-throughput, low-cost tools for monitoring the folding and unfolding transitions of G-rich sequences would provide an enabling technology for accelerating the prototyping of synthetic biological systems and for accelerating design-build-test cycles. Here, we show that unfolding transitions of a range of G-quadruplex-forming DNA sequences can be monitored in a FRET-like format using DNA sequences that possess only a single dye label, with no quencher. These quencher-free assays can be performed at low cost, with both cost and lead times ca. 1 order of magnitude lower than FRET-labeled strands. Thus, quencher-free secondary structure monitoring promises to be a valuable tool for the testing and development of synthetic biology systems employing G-quadruplexes.

  PublisherDOI

• Controlled exchange of protein and nucleic acid signals from and between synthetic minimal cells

  Cell Systems · 2024-01-01 · 15 citations

  article
  PublisherDOI

• Sequential gentle hydration increases encapsulation in model protocells

  Discover Life · 2024-05-13 · 4 citations

  articleOpen access

  Small, spherical vesicles are a widely used chassis for the formation of model protocells and investigating the beginning of compartmentalized evolution. Various methods exist for their preparation, with one of the most common approaches being gentle hydration, where thin layers of lipids are hydrated with aqueous solutions and gently agitated to form vesicles. An important benefit to gentle hydration is that the method produces vesicles without introducing any organic contaminants, such as mineral oil, into the lipid bilayer. However, compared to other methods of liposome formation, gentle hydration is much less efficient at encapsulating aqueous cargo. Improving the encapsulation efficiency of gentle hydration would be of broad use for medicine, biotechnology, and protocell research. Here, we describe a method of sequentially hydrating lipid thin films to increase encapsulation efficiency. We demonstrate that sequential gentle hydration significantly improves encapsulation of water-soluble cargo compared to the traditional method, and that this improved efficiency is dependent on buffer composition. Similarly, we also demonstrate how this method can be used to increase concentrations of oleic acid, a fatty acid commonly used in origins of life research, to improve the formation of vesicles in aqueous buffer.

  PublisherOA PDFDOI

• PACRAT: Pathogen detection with aptamer-detected cascaded recombinase polymerase amplification-in vitro transcription

  RNA · 2024-04-18

  articleOpen accessSenior author

  The SARS-CoV-2 pandemic underscored the need for early, rapid, and widespread pathogen detection tests that are readily accessible. Many existing rapid isothermal detection methods employ the recombinase polymerase amplification (RPA), which exhibits PCR-like sensitivity, specificity, and even higher speed. However, coupling RPA to other enzymatic reactions has proven difficult. For the first time, we demonstrate that with tuning of buffer conditions and optimization of reagent concentrations, RPA can be cascaded into an in vitro transcription reaction, enabling detection using fluorescent aptamers in a one-pot reaction. We show that this reaction, which we term PACRAT (Pathogen detection with Aptamer-detected Cascaded Recombinase polymerase Amplification-in vitro Transcription) can be used to detect SARS-CoV-2 with single-copy detection limits and 10-minute detection times. Further demonstrating the utility of our one-pot, cascaded amplification system, we show PACRAT can be employed for multiplexed detection of the pathogens SARS-CoV-2 and E. coli, along with multiplexed detection of two variants of SARS-CoV-2.

  PublisherOA PDFDOI

• Quencher-free fluorescence monitoring of G-quadruplex folding

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-01-31 · 1 citations

  preprintOpen accessSenior authorCorresponding

  Abstract Guanine-rich sequences exhibit a high degree of polymorphism and can form single-stranded, Watson-Crick duplex, and four-stranded G-quadruplex structures. These sequences have found a wide range of uses in synthetic biology applications, arising in part from their structural plasticity. High-throughput, low-cost tools for monitoring the folding and unfolding transitions of G-rich sequences would provide an enabling technology for accelerating prototyping of synthetic biological systems and for accelerating design-build-test cycles. Here, we show that unfolding transitions of a range of G-quadruplex-forming DNA sequences can be monitored in a FRET-like format using DNA sequences that possess only a single dye label, with no quencher. These quencher-free assays can be performed at low cost, with both cost and lead times ca. 1 order of magnitude lower than FRET-labeled strands. Thus, quencher-free secondary structure monitoring promises to be a valuable tool for testing and development of synthetic biology systems employing G-quadruplexes.

  PublisherOA PDFDOI

Frequent coauthors

• Katarzyna P. Adamala

  University of Minnesota

  70 shared

• Jack W. Szostak

  46 shared

• Nathaniel J. Gaut

  University of Minnesota

  22 shared

• Nicholas V. Hud

  Georgia Institute of Technology

  18 shared

• Christopher Deich

  University of Minnesota

  14 shared

• Lauren M. Aufdembrink

  University of Minnesota System

  13 shared

• Jia Sheng

  University at Albany, State University of New York

  13 shared

• Lin Jin

  Wuhan Institute of Bioengineering

  12 shared