Publications by authors named "Andrew Ellington"

330 Publications

Nanoaggregate-forming lipid-conjugated AS1411 aptamer as a promising tumor-targeted delivery system of anticancer agents in vitro.

Nanomedicine 2021 08 24;36:102429. Epub 2021 Jun 24.

CICS-UBI-Centro de Investigação em Ciências da Saúde, Universidade da Beira Interior, Covilhã, Portugal. Electronic address:

Nanoparticles offer targeted delivery of drugs with minimal toxicity to surrounding healthy tissue and have great potential in the management of human papillomavirus (HPV)-related diseases. We synthesized lipid-modified AS1411 aptamers capable of forming nanoaggregates in solution containing Mg. The nanoaggregates presented suitable properties for pharmaceutical applications such as small size (100 nm), negative charge, and drug release. The nanoaggregates were loaded with acridine orange derivative C for its specific delivery into cervical cancer cell lines and HPV-positive tissue biopsies. This improved inhibition of HeLa proliferation and cell uptake without significantly affecting healthy cells. Finally, the nanoaggregates were incorporated in a gel formulation with promising tissue retention properties aiming at developing a local delivery strategy of the nanoaggregates in the female genital tract. Collectively, these findings suggest that the nanoformulation protocol has great potential for the delivery of both anticancer and antiviral agents, becoming a novel modality for cervical cancer management.
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http://dx.doi.org/10.1016/j.nano.2021.102429DOI Listing
August 2021

Genetic transformation technologies for the common dandelion, Taraxacum officinale.

Plant Methods 2021 Jun 9;17(1):59. Epub 2021 Jun 9.

Department of Molecular Biosciences, College of Natural Sciences, The University of Texas at Austin, Austin, TX, 78712, USA.

Background: Taraxacum officinale, or the common dandelion, is a widespread perennial species recognized worldwide as a common lawn and garden weed. Common dandelion is also cultivated for use in teas, as edible greens, and for use in traditional medicine. It produces latex and is closely related to the Russian dandelion, T. kok-saghyz, which is being developed as a rubber crop. Additionally, the vast majority of extant common dandelions reproduce asexually through apomictically derived seeds- an important goal for many major crops in modern agriculture. As such, there is increasing interest in the molecular control of important pathways as well as basic molecular biology and reproduction of common dandelion.

Results: Here we present an improved Agrobacterium-based genetic transformation and regeneration protocol, a protocol for generation and transformation of protoplasts using free DNA, and a protocol for leaf Agrobacterium infiltration for transient gene expression. These protocols use easily obtainable leaf explants from soil-grown plants and reagents common to most molecular plant laboratories. We show that common markers used in many plant transformation systems function as expected in common dandelion including fluorescent proteins, GUS, and anthocyanin regulation, as well as resistance to kanamycin, Basta, and hygromycin.

Conclusion: Reproducible, stable and transient transformation methods are presented that will allow for needed molecular structure and function studies of genes and proteins in T. officinale.
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http://dx.doi.org/10.1186/s13007-021-00760-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8191202PMC
June 2021

The Grayness of the Origin of Life.

Life (Basel) 2021 May 29;11(6). Epub 2021 May 29.

Department of Biology, Georgetown University, Washington, DC 20057, USA.

In the search for life beyond Earth, distinguishing the living from the non-living is paramount. However, this distinction is often elusive, as the origin of life is likely a stepwise evolutionary process, not a singular event. Regardless of the favored origin of life model, an inherent "grayness" blurs the theorized threshold defining life. Here, we explore the ambiguities between the biotic and the abiotic at the origin of life. The role of grayness extends into later transitions as well. By recognizing the limitations posed by grayness, life detection researchers will be better able to develop methods sensitive to prebiotic chemical systems and life with alternative biochemistries.
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http://dx.doi.org/10.3390/life11060498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8226951PMC
May 2021

Producing molecular biology reagents without purification.

PLoS One 2021 1;16(6):e0252507. Epub 2021 Jun 1.

Department of Molecular Biosciences, College of Natural Sciences, The University of Texas at Austin, Austin, Texas, United States of America.

We recently developed 'cellular' reagents-lyophilized bacteria overexpressing proteins of interest-that can replace commercial pure enzymes in typical diagnostic and molecular biology reactions. To make cellular reagent technology widely accessible and amenable to local production with minimal instrumentation, we now report a significantly simplified method for preparing cellular reagents that requires only a common bacterial incubator to grow and subsequently dry enzyme-expressing bacteria at 37°C with the aid of inexpensive chemical desiccants. We demonstrate application of such dried cellular reagents in common molecular and synthetic biology processes, such as PCR, qPCR, reverse transcription, isothermal amplification, and Golden Gate DNA assembly, in building easy-to-use testing kits, and in rapid reagent production for meeting extraordinary diagnostic demands such as those being faced in the ongoing SARS-CoV-2 pandemic. Furthermore, we demonstrate feasibility of local production by successfully implementing this minimized procedure and preparing cellular reagents in several countries, including the United Kingdom, Cameroon, and Ghana. Our results demonstrate possibilities for readily scalable local and distributed reagent production, and further instantiate the opportunities available via synthetic biology in general.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0252507PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8168896PMC
June 2021

High-Surety Isothermal Amplification and Detection of SARS-CoV-2.

mSphere 2021 05 19;6(3). Epub 2021 May 19.

Department of Molecular Biosciences, College of Natural Sciences, The University of Texas at Austin, Austin, Texas, USA

Isothermal nucleic acid amplification tests (iNATs), such as loop-mediated isothermal amplification (LAMP), are good alternatives to PCR-based amplification assays, especially for point-of-care and low-resource use, in part because they can be carried out with relatively simple instrumentation. However, iNATs can often generate spurious amplicons, especially in the absence of target sequences, resulting in false-positive results. This is especially true if signals are based on non-sequence-specific probes, such as intercalating dyes or pH changes. In addition, pathogens often prove to be moving, evolving targets and can accumulate mutations that will lead to inefficient primer binding and thus false-negative results. Multiplex assays targeting different regions of the analyte and logical signal readout using sequence-specific probes can help to reduce both false negatives and false positives. Here, we describe rapid conversion of three previously described SARS-CoV-2 LAMP assays that relied on a non-sequence-specific readout into individual and multiplex one-pot assays that can be visually read using sequence-specific oligonucleotide strand exchange (OSD) probes. We describe both fluorescence-based and Boolean logic-gated colorimetric lateral flow readout methods and demonstrate detection of SARS-CoV-2 virions in crude human saliva. One of the key approaches to treatment and control of infectious diseases, such as COVID-19, is accurate and rapid diagnostics that is widely deployable in a timely and scalable manner. To achieve this, it is essential to go beyond the traditional gold standard of quantitative PCR (qPCR) that is often faced with difficulties in scaling due to the complexity of infrastructure and human resource requirements. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), have been long pursued as ideal, low-tech alternatives for rapid, portable testing. However, isothermal approaches often suffer from false signals due to employment of nonspecific readout methods. We describe general principles for rapidly converting nonspecifically read LAMP assays into assays that are read in a sequence-specific manner by using oligonucleotide strand displacement (OSD) probes. We also demonstrate that inclusion of OSD probes in LAMP assays maintains the simplicity of one-pot assays and a visual yes/no readout by using fluorescence or colorimetric lateral-flow dipsticks while providing accurate sequence-specific readout and the ability to logically query multiplex amplicons for redundancy or copresence. These principles not only yielded high-surety isothermal assays for SARS-CoV-2 but might also aid in the design of more sophisticated molecular assays for other analytes.
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http://dx.doi.org/10.1128/mSphere.00911-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8265673PMC
May 2021

Minimizing Leakage in Stacked Strand Exchange Amplification Circuits.

ACS Synth Biol 2021 06 18;10(6):1277-1283. Epub 2021 May 18.

Department of Molecular Biosciences, Center for Systems and Synthetic Biology, College of Natural Sciences, The University of Texas at Austin, Austin, Texas 78712, United States.

Signal amplification is ubiquitous in biology and engineering. Protein enzymes, such as DNA polymerases, can routinely achieve >10-fold signal increase, making them powerful tools for signal enhancement. Considerable signal amplification can also be achieved using nonenzymatic, cascaded nucleic acid strand exchange reactions. However, the practical application of such kinetically trapped circuits has so far proven difficult due to uncatalyzed leakage of the cascade. We now demonstrate that strategically positioned mismatches between circuit components can reduce unprogrammed hybridization reactions and therefore greatly diminish leakage. In consequence, we were able to synthesize a three-layer catalytic hairpin assembly cascade that could operate in a single tube and that yielded 3.7 × 10-fold signal amplification in only 4 h, a greatly improved performance relative to previous cascades. This advance should facilitate the implementation of nonenzymatic signal amplification in molecular diagnostics, as well as inform the design of a wide variety of increasingly intricate nucleic acid computation circuits.
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http://dx.doi.org/10.1021/acssynbio.0c00615DOI Listing
June 2021

Guiding Ethical Principles in Engineering Biology Research.

ACS Synth Biol 2021 05 12;10(5):907-910. Epub 2021 May 12.

Engineering Biology Research Consortium, Emeryville, California 94608, United States.

Engineering biology is being applied toward solving or mitigating some of the greatest challenges facing society. As with many other rapidly advancing technologies, the development of these powerful tools must be considered in the context of ethical uses for personal, societal, and/or environmental advancement. Researchers have a responsibility to consider the diverse outcomes that may result from the knowledge and innovation they contribute to the field. Together, we developed a Statement of Ethics in Engineering Biology Research to guide researchers as they incorporate the consideration of long-term ethical implications of their work into every phase of the research lifecycle. Herein, we present and contextualize this Statement of Ethics and its six guiding principles. Our goal is to facilitate ongoing reflection and collaboration among technical researchers, social scientists, policy makers, and other stakeholders to support best outcomes in engineering biology innovation and development.
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http://dx.doi.org/10.1021/acssynbio.1c00129DOI Listing
May 2021

Directed Evolution of an Improved Aminoacyl-tRNA Synthetase for Incorporation of L-3,4-Dihydroxyphenylalanine (L-DOPA).

Angew Chem Int Ed Engl 2021 06 24;60(27):14811-14816. Epub 2021 May 24.

Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA.

The catechol group of 3,4-dihydroxyphenylalanine (L-DOPA) derived from L-tyrosine oxidation is a key post-translational modification (PTM) in many protein biomaterials and has potential as a bioorthogonal handle for precision protein conjugation applications such as antibody-drug conjugates. Despite this potential, indiscriminate enzymatic modification of exposed tyrosine residues or complete replacement of tyrosine using auxotrophic hosts remains the preferred method of introducing the catechol moiety into proteins, which precludes many protein engineering applications. We have developed new orthogonal translation machinery to site-specifically incorporate L-DOPA into recombinant proteins and a new fluorescent biosensor to selectively monitor L-DOPA incorporation in vivo. We show simultaneous biosynthesis and incorporation of L-DOPA and apply this translation machinery to engineer a novel metalloprotein containing a DOPA-Fe chromophore.
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http://dx.doi.org/10.1002/anie.202100579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8217333PMC
June 2021

Synthetic repertoires derived from convalescent COVID-19 patients enable discovery of SARS-CoV-2 neutralizing antibodies and a novel quaternary binding modality.

bioRxiv 2021 Apr 9. Epub 2021 Apr 9.

The ongoing evolution of SARS-CoV-2 into more easily transmissible and infectious variants has sparked concern over the continued effectiveness of existing therapeutic antibodies and vaccines. Hence, together with increased genomic surveillance, methods to rapidly develop and assess effective interventions are critically needed. Here we report the discovery of SARS-CoV-2 neutralizing antibodies isolated from COVID-19 patients using a high-throughput platform. Antibodies were identified from unpaired donor B-cell and serum repertoires using yeast surface display, proteomics, and public light chain screening. Cryo-EM and functional characterization of the antibodies identified N3-1, an antibody that binds avidly (K = 68 pM) to the receptor binding domain (RBD) of the spike protein and robustly neutralizes the virus . This antibody likely binds all three RBDs of the trimeric spike protein with a single IgG. Importantly, N3-1 equivalently binds spike proteins from emerging SARS-CoV-2 variants of concern, neutralizes UK variant B.1.1.7, and binds SARS-CoV spike with nanomolar affinity. Taken together, the strategies described herein will prove broadly applicable in interrogating adaptive immunity and developing rapid response biological countermeasures to emerging pathogens.
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http://dx.doi.org/10.1101/2021.04.07.438849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8043448PMC
April 2021

Ribosome-mediated incorporation of fluorescent amino acids into peptides in vitro.

Chem Commun (Camb) 2021 Mar 16;57(21):2661-2664. Epub 2021 Feb 16.

Department of Chemical and Biological Engineering and Center for Synthetic Biology, Northwestern University, Evanston, IL 60208, USA.

We report the design, chemical synthesis, and flexizyme-catalyzed transfer RNA (tRNA) acylation of a variety of fluorescent amino acids (FAAs). The fluorescent groups include pyrene, coumarin, nitrobenzoxadiazole, and fluorescein variants. We further demonstrate site-specific incorporation of the FAAs into peptides by the ribosome in vitro through genetic code reprogramming.
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http://dx.doi.org/10.1039/d0cc07740bDOI Listing
March 2021

Hurdling and Hurtling Toward New Genetic Codes.

ACS Cent Sci 2021 Jan 10;7(1):7-10. Epub 2020 Dec 10.

Center for Systems and Synthetic Biology University of Texas at Austin Austin, Texas 78712, United States.

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http://dx.doi.org/10.1021/acscentsci.0c01549DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7844845PMC
January 2021

One-Enzyme Reverse Transcription qPCR Using Taq DNA Polymerase.

Biochemistry 2020 Dec 4;59(49):4638-4645. Epub 2020 Dec 4.

Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, Texas 78712, United States.

Taq DNA polymerase, one of the first thermostable DNA polymerases to be discovered, has been typecast as a DNA-dependent DNA polymerase commonly employed for PCR. However, Taq polymerase belongs to the same DNA polymerase superfamily as the Molony murine leukemia virus reverse transcriptase and has in the past been shown to possess reverse transcriptase activity. We report optimized buffer and salt compositions that promote the reverse transcriptase activity of Taq DNA polymerase and thereby allow it to be used as the sole enzyme in TaqMan RT-qPCRs. We demonstrate the utility of Taq-alone RT-qPCRs by executing CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays that could detect as few as 2 copies/μL of input viral genomic RNA.
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http://dx.doi.org/10.1021/acs.biochem.0c00778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7757722PMC
December 2020

Discovery of Novel Gain-of-Function Mutations Guided by Structure-Based Deep Learning.

ACS Synth Biol 2020 11 16;9(11):2927-2935. Epub 2020 Oct 16.

Center for Systems and Synthetic Biology, The Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712, United States.

Despite the promise of deep learning accelerated protein engineering, examples of such improved proteins are scarce. Here we report that a 3D convolutional neural network trained to associate amino acids with neighboring chemical microenvironments can guide identification of novel gain-of-function mutations that are not predicted by energetics-based approaches. Amalgamation of these mutations improved protein function across three diverse proteins by at least 5-fold. Furthermore, this model provides a means to interrogate the chemical space within protein microenvironments and identify specific chemical interactions that contribute to the gain-of-function phenotypes resulting from individual mutations.
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http://dx.doi.org/10.1021/acssynbio.0c00345DOI Listing
November 2020

ΦX174 Attenuation by Whole-Genome Codon Deoptimization.

Genome Biol Evol 2021 02;13(2)

Department of Biological Science, University of Idaho.

Natural selection acting on synonymous mutations in protein-coding genes influences genome composition and evolution. In viruses, introducing synonymous mutations in genes encoding structural proteins can drastically reduce viral growth, providing a means to generate potent, live-attenuated vaccine candidates. However, an improved understanding of what compositional features are under selection and how combinations of synonymous mutations affect viral growth is needed to predictably attenuate viruses and make them resistant to reversion. We systematically recoded all nonoverlapping genes of the bacteriophage ΦX174 with codons rarely used in its Escherichia coli host. The fitness of recombinant viruses decreases as additional deoptimizing mutations are made to the genome, although not always linearly, and not consistently across genes. Combining deoptimizing mutations may reduce viral fitness more or less than expected from the effect size of the constituent mutations and we point out difficulties in untangling correlated compositional features. We test our model by optimizing the same genes and find that the relationship between codon usage and fitness does not hold for optimization, suggesting that wild-type ΦX174 is at a fitness optimum. This work highlights the need to better understand how selection acts on patterns of synonymous codon usage across the genome and provides a convenient system to investigate the genetic determinants of virulence.
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http://dx.doi.org/10.1093/gbe/evaa214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7881332PMC
February 2021

Emulsion-based directed evolution of enzymes and proteins in yeast.

Methods Enzymol 2020 13;643:87-110. Epub 2020 May 13.

Center for Systems and Synthetic Biology, The Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, United States. Electronic address:

Emulsion-based selections are a unique type of directed evolution method that overcome common bottlenecks associated with purely in vivo selections. For example, emulsions including cell-free translation machinery can be useful for expression of toxic genes. However, not all cell types can efficiently produce protein in vitro, for example, the eukaryotic microbe Saccharomyces cerevisiae. compartmentalized self replication (CSR) and compartmentalized partnered replication (CPR) are two emulsion-based selection schemes that leverage the advantages of both in vivo and in vitro selections by compartmentalizing cells in water-in-oil droplets. Previous implementations of these methods utilized bacterial hosts, which has limited the technology to the directed evolution of proteins that can be heterologously expressed in prokaryotic systems. To expand the repertoire of targets that can be evolved, we have adapted emulsion-based PCR selections to be compatible with a eukaryotic host.
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http://dx.doi.org/10.1016/bs.mie.2020.04.053DOI Listing
June 2021

How a B family DNA polymerase has been evolved to copy RNA.

Proc Natl Acad Sci U S A 2020 09 17;117(35):21274-21280. Epub 2020 Aug 17.

Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892;

We report here crystal structures of a reverse transcriptase RTX, which was evolved in vitro from the B family polymerase KOD, in complex with either a DNA duplex or an RNA-DNA hybrid. Compared with the apo, binary, and ternary complex structures of the original KOD polymerase, the 16 substitutions that result in the function of copying RNA to DNA do not change the overall protein structure. Only six substitutions occur at the substrate-binding surface, and the others change domain-domain interfaces in the polymerase to enable RNA-DNA hybrid binding and reverse transcription. Most notably, F587L at the Palm and Thumb interface stabilizes the open and apo conformation of the Thumb. The intrinsically flexible Thumb domain seems to play a major role in accommodating the RNA-DNA hybrid product distal to the active site. This is reminiscent of naturally occurring RNA-dependent DNA polymerases, including telomerase, which have a dramatically augmented Thumb domain, and of reverse transcriptase, which extends its Thumb with the RNase H domain.
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http://dx.doi.org/10.1073/pnas.2009415117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474658PMC
September 2020

Comparison of media and standards for SARS-CoV-2 RT-qPCR without prior RNA preparation.

medRxiv 2020 Sep 17. Epub 2020 Sep 17.

Department of Molecular Biosciences, College of Natural Sciences, The University of Texas at Austin, Austin, TX 78712, USA.

Since the emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there have been demands on the testing infrastructure that have strained testing capacity. As a simplification of method, we confirm the efficacy of RNA extraction-free RT-qPCR and saline as an alternative patient sample storage buffer. In addition, amongst potential reagent shortages, it has sometimes been difficult to obtain inactivated viral particles. We have therefore also characterized armored SARS-CoV-2 RNA from Asuragen as an alternative diagnostic standard to ATCC genomic SARS-CoV-2 RNA and heat inactivated virions and provide guidelines for its use in RT-qPCR.
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http://dx.doi.org/10.1101/2020.08.01.20166173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418746PMC
September 2020

A facile technology for the high-throughput sequencing of the paired VH:VL and TCRβ:TCRα repertoires.

Sci Adv 2020 Apr 22;6(17):eaay9093. Epub 2020 Apr 22.

Department of Chemical Engineering, University of Texas at Austin, Austin, TX, USA.

Natively paired sequencing (NPS) of B cell receptors [variable heavy (VH) and light (VL)] and T cell receptors (TCRb and TCRa) is essential for the understanding of adaptive immunity in health and disease. Despite many recent technical advances, determining the VH:VL or TCRb:a repertoire with high accuracy and throughput remains challenging. We discovered that the recently engineered xenopolymerase, RTX, is exceptionally resistant to cell lysate inhibition in single-cell emulsion droplets. We capitalized on the characteristics of this enzyme to develop a simple, rapid, and inexpensive in-droplet overlap extension reverse transcription polymerase chain reaction method for NPS not requiring microfluidics or other specialized equipment. Using this technique, we obtained high yields (5000 to >20,000 per sample) of paired VH:VL or TCRb:a clonotypes at low cost. As a demonstration, we performed NPS on peripheral blood plasmablasts and T follicular helper cells following seasonal influenza vaccination and discovered high-affinity influenza-specific antibodies and TCRb:a.
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http://dx.doi.org/10.1126/sciadv.aay9093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176429PMC
April 2020

Ribosomal incorporation of cyclic β-amino acids into peptides using in vitro translation.

Chem Commun (Camb) 2020 May;56(42):5597-5600

Department of Chemical and Biological Engineering and Center for Synthetic Biology, Northwestern University, Evanston, IL 60208, USA.

We demonstrate in vitro incorporation of cyclic β-amino acids into peptides by the ribosome through genetic code reprogramming. Further, we show that incorporation efficiency can be increased through the addition of elongation factor P.
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http://dx.doi.org/10.1039/d0cc02121kDOI Listing
May 2020

Computer-based Engineering of Thermostabilized Antibody Fragments.

AIChE J 2020 Mar 19;66(3). Epub 2019 Nov 19.

Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712.

We used the molecular modeling program Rosetta to identify clusters of amino acid substitutions in antibody fragments (scFvs and scAbs) that improve global protein stability and resistance to thermal deactivation. Using this methodology, we increased the melting temperature (T) and resistance to heat treatment of an antibody fragment that binds to the hemagglutinin protein (anti-HA33). Two designed antibody fragment variants with two amino acid replacement clusters, designed to stabilize local regions, were shown to have both higher T compared to the parental scFv and importantly, to retain full antigen binding activity after 2 hours of incubation at 70 °C. The crystal structure of one thermostabilized scFv variants was solved at 1.6 Å and shown to be in close agreement with the RosettaAntibody model prediction.
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http://dx.doi.org/10.1002/aic.16864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181397PMC
March 2020

A highly stable RNA aptamer probe for the retinoblastoma protein in live cells.

Chem Sci 2020 Apr 21;11(17):4467-4474. Epub 2020 Apr 21.

Department of Chemistry, Imperial College London 80 Wood Lane W12 0BZ UK

Although RNA aptamers can show comparable or better specificity and affinity to antibodies and have the advantage of being able to access different live cell compartments, they are often much less stable . We report here the first aptamer that binds human retinoblastoma protein (RB) and is stable in live cells. RB is both a key protein in cell cycle control and also a tumour suppressor. The aptamer was selected from an RNA library against a unique 12-residue helical peptide derived from RB rather than the whole protein molecule. It binds RB with high affinity ( = 5.1 ± 0.1 nM) and is a putative RNA G-quadruplex structure formed by an 18-nucleotide sequence (18E16 - GGA GGG UGG AGG GAA GGG), which may account for its high stability. Confocal fluorescence microscopy of live cells transfected with the aptamer shows it is stable intracellularly and efficient in entering the nucleus where an analogous antibody was inaccessible. The findings demonstrate this aptamer is an advanced probe for RB in live cell applications.
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http://dx.doi.org/10.1039/d0sc01613fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8159449PMC
April 2020

Correction: Directed evolution of gold nanoparticle delivery to cells.

Chem Commun (Camb) 2020 04 3;56(31):4368. Epub 2020 Apr 3.

Department of Chemistry and Biochemistry, Institute for Cell and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.

Correction for 'Directed evolution of gold nanoparticle delivery to cells' by Na Li et al., Chem. Commun., 2010, 46, 392-394.
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http://dx.doi.org/10.1039/d0cc90149kDOI Listing
April 2020

Dynamic Programming of a DNA Walker Controlled by Protons.

ACS Nano 2020 04 17;14(4):4007-4013. Epub 2020 Mar 17.

Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea.

We have now constructed a four-legged DNA walker based on toehold exchange reactions whose movement is controlled by alternating pH changes. A well-characterized, pH-responsive CG-C triplex DNA was embedded into a tetrameric catalytic hairpin assembly (CHA) walker. The proton-controlled walker could autonomously move on otherwise unprogrammed microparticles surface, and the walking rate and steps of walking were efficiently controlled by pH. The starting and stopping of the walker, and its association and dissociation from the microparticles, could also be dynamically controlled by pH. The simple, programmable, and robust nature of this proton-controlled walker now provides the impetus for the development of a wide variety of more practical nanomachines.
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http://dx.doi.org/10.1021/acsnano.9b08166DOI Listing
April 2020

Site-specific 5-hydroxytryptophan incorporation into apolipoprotein A-I impairs cholesterol efflux activity and high-density lipoprotein biogenesis.

J Biol Chem 2020 04 25;295(15):4836-4848. Epub 2020 Feb 25.

Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195

Apolipoprotein A-I (apoA-I) is the major protein constituent of high-density lipoprotein (HDL) and a target of myeloperoxidase-dependent oxidation in the artery wall. In atherosclerotic lesions, apoA-I exhibits marked oxidative modifications at multiple sites, including Trp Site-specific mutagenesis studies have suggested, but have not conclusively shown, that oxidative modification of Trp of apoA-I impairs many atheroprotective properties of this lipoprotein. Herein, we used genetic code expansion technology with an engineered tryptophanyl tRNA-synthetase (Trp-RS):suppressor tRNA pair to insert the noncanonical amino acid 5-hydroxytryptophan (5-OHTrp) at position 72 in recombinant human apoA-I and confirmed site-specific incorporation utilizing MS. In functional characterization studies, 5-OHTrp apoA-I (compared with WT apoA-I) exhibited reduced ABC subfamily A member 1 (ABCA1)-dependent cholesterol acceptor activity (41.73 ± 6.57% inhibition; < 0.01). Additionally, 5-OHTrp apoA-I displayed increased activation and stabilization of paraoxonase 1 (PON1) activity (μmol/min/mg) when compared with WT apoA-I and comparable PON1 activation/stabilization compared with reconstituted HDL (WT apoA-I, 1.92 ± 0.04; 5-OHTrp apoA-I, 2.35 ± 0.0; and HDL, 2.33 ± 0.1; < 0.001, < 0.001, and < 0.001, respectively). Following injection into apoA-I-deficient mice, 5-OHTrp apoA-I reached plasma levels comparable with those of native apoA-I yet exhibited significantly reduced (48%; < 0.01) lipidation and evidence of HDL biogenesis. Collectively, these findings unequivocally reveal that site-specific oxidative modification of apoA-I via 5-OHTrp at Trp impairs cholesterol efflux and the rate-limiting step of HDL biogenesis both and .
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http://dx.doi.org/10.1074/jbc.RA119.012092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152772PMC
April 2020

Engineered symbionts activate honey bee immunity and limit pathogens.

Science 2020 01 30;367(6477):573-576. Epub 2020 Jan 30.

Department of Integrative Biology, The University of Texas at Austin, Austin, TX 78712, USA.

Honey bees are essential pollinators threatened by colony losses linked to the spread of parasites and pathogens. Here, we report a new approach for manipulating bee gene expression and protecting bee health. We engineered a symbiotic bee gut bacterium, , to induce eukaryotic RNA interference (RNAi) immune responses. We show that engineered can stably recolonize bees and produce double-stranded RNA to activate RNAi and repress host gene expression, thereby altering bee physiology, behavior, and growth. We used this approach to improve bee survival after a viral challenge, and we show that engineered can kill parasitic mites by triggering the mite RNAi response. This symbiont-mediated RNAi approach is a tool for studying bee functional genomics and potentially for safeguarding bee health.
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http://dx.doi.org/10.1126/science.aax9039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556694PMC
January 2020

Oligonucleotide-functionalized hydrogels for sustained release of small molecule (aptamer) therapeutics.

Acta Biomater 2020 01 21;102:315-325. Epub 2019 Nov 21.

J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida, 1275 Center Dr, Gainesville, FL 32615, USA. Electronic address:

Natural and synthetic hydrogels have been widely investigated as biomaterial scaffolds to promote tissue repair and regeneration. Nevertheless, the scaffold alone is often insufficient to drive new tissue growth, instead requiring continuous delivery of therapeutics, such as proteins or other biomolecules that work in concert with structural support provided by the scaffold. However, because of the high-water content, hydrogels tend to be permeable and cause rapid release of the encapsulated drug, which could lead to serious complications from local overdose and may result in the significant waste of encapsulated therapeutic(s). To this end, we designed an oligonucleotide-functionalized hydrogel that can provide sustained and controlled delivery of therapeutics for up to 4 weeks. To prove this concept, we successfully achieved sustained release (for over 28 days) of model anti-Nogo receptor (anti-NgR) RNA aptamer from oligonucleotide-functionalized hyaluronic acid-based hydrogel by changing the complementarity between the short antisense sequences and the aptamer. Furthermore, the released aptamer successfully blocked neuro-inhibitory effects of myelin-derived inhibitors and promoted neurite outgrowth from rat dorsal root ganglia in vitro. Because antisense sequences can be designed to bind to proteins, peptides, and aptamer, our oligonucleotide-functionalized hydrogel offers a promising therapeutic delivery system to obtain controlled release (both bolus and sustained) of various therapeutics for the treatment of complex diseases and injury models, such as spinal cord injury. STATEMENT OF SIGNIFICANCE: Producing a therapeutic effect often requires the administration of multiple injections with high dosages. This regimen causes discomfort to the patient and raises cost of treatment. Additionally, systemic delivery of therapeutics often results in adverse effects; therefore, local delivery at the site of injury is desirable. Therefore, in this study, we designed an oligonucleotide-functionalized biomaterial platform using ssDNA oligonucleotides (immobile species) as antisense sequences to increase residence time and fine-tune the release of anti-nogo receptor aptamer (mobile species) for spinal cord injury application. Because antisense sequences can be designed to bind proteins, peptides, and aptamer, our hydrogel offers a promising delivery system to obtain controlled release of various therapeutics for the treatment of complex diseases and injury models.
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http://dx.doi.org/10.1016/j.actbio.2019.11.037DOI Listing
January 2020

Expanding the limits of the second genetic code with ribozymes.

Nat Commun 2019 11 8;10(1):5097. Epub 2019 Nov 8.

Department of Chemical and Biological Engineering, Northwestern University, Evanston, 60208, IL, USA.

The site-specific incorporation of noncanonical monomers into polypeptides through genetic code reprogramming permits synthesis of bio-based products that extend beyond natural limits. To better enable such efforts, flexizymes (transfer RNA (tRNA) synthetase-like ribozymes that recognize synthetic leaving groups) have been used to expand the scope of chemical substrates for ribosome-directed polymerization. The development of design rules for flexizyme-catalyzed acylation should allow scalable and rational expansion of genetic code reprogramming. Here we report the systematic synthesis of 37 substrates based on 4 chemically diverse scaffolds (phenylalanine, benzoic acid, heteroaromatic, and aliphatic monomers) with different electronic and steric factors. Of these substrates, 32 were acylated onto tRNA and incorporated into peptides by in vitro translation. Based on the design rules derived from this expanded alphabet, we successfully predicted the acylation of 6 additional monomers that could uniquely be incorporated into peptides and direct N-terminal incorporation of an aldehyde group for orthogonal bioconjugation reactions.
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http://dx.doi.org/10.1038/s41467-019-12916-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6841967PMC
November 2019

Synthetic GPCRs and signal transduction cascades.

Emerg Top Life Sci 2019 Nov;3(5):609-614

Center for Systems and Synthetic Biology and Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, U.S.A.

G protein-coupled receptors (GPCRs) are a large and diverse group of membrane proteins that constitute over 30% of FDA approved drug targets. Despite their importance, much remains unknown about GPCR signaling at a system's level. Efforts to engineer receptors with orthogonal components have attempted to provide tools to parse signaling and resultant phenotypes. Recent advances in synthetic biology provide opportunities to engineer receptors at scale and with additional properties that could further inform GPCR biology at a system's level, and enhance the ability to engineer complex signal transduction.
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http://dx.doi.org/10.1042/ETLS20190035DOI Listing
November 2019

Single-Molecule Mechanistic Study of Enzyme Hysteresis.

ACS Cent Sci 2019 Oct 24;5(10):1691-1698. Epub 2019 Sep 24.

Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, United States.

Hysteresis is an important feature of enzyme-catalyzed reactions, as it reflects the influence of enzyme regulation in the presence of ligands such as substrates or allosteric molecules. In typical kinetic studies of enzyme activity, hysteretic behavior is observed as a "lag" or "burst" in the time course of the catalyzed reaction. These lags and bursts are due to the relatively slow transition from one state to another state of the enzyme molecule, with different states having different kinetic properties. However, it is difficult to understand the underlying mechanism of hysteresis by observing bulk reactions because the different enzyme molecules in the population behave stochastically. In this work, we studied the hysteretic behavior of mutant β-glucuronidase (GUS) using a high-throughput single-molecule array platform and investigated the effect of thermal treatment on the hysteresis.
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http://dx.doi.org/10.1021/acscentsci.9b00718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813718PMC
October 2019
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