Biology subjects

Shepherd, T. R.

Publications and source records attributed to Shepherd, T. R..

7 recordsLinked to original sources

bioRxiv2022

3D RNA-scaffolded wireframe origami

Hybrid RNA:DNA origami, in which a long RNA scaffold strand is folded into a target nanostructure via thermal annealing with complementary DNA oligos, has only been explored to a limited extent despite its unique potential for biomedical delivery of mRNA, tertiary structure characterization of long RNAs, and fabrication of artificial ribozymes. Here, we investigate design principles of wireframe RNA-scaffolded origami in three dimensions rendered as polyhedra composed of dual-duplex edges. We computationally designed, fabricated, and characterized tetrahedra folded from an EGFP-encoding messenger RNA and de Bruijn sequences, an octahedron folded with M13 transcript RNA, and an octahedron and pentagonal bipyramids folded with 23S ribosomal RNA, demonstrating the ability to make diverse polyhedral shapes with distinct structural and functional RNA scaffolds. We characterized secondary and tertiary structures using dimethyl sulfate mutational profiling and cryo-electron microscopy, revealing for the first time insight into both global and local, base-level structures of origami. Our top-down sequence design strategy enables the use of long RNAs as functional scaffolds for complex wireframe origami.

bioengineering↗

bioRxiv2021

A framework for evaluating edited cell libraries created by massively parallel genome engineering

Genome engineering methodologies are transforming biological research and discovery. Approaches based on CRISPR technology have been broadly adopted and there is growing interest in the generation of massively parallel edited cell libraries. Comparing the libraries generated by these varying approaches is challenging and researchers lack a common framework for defining and assessing the characteristics of these libraries. Here we describe a framework for evaluating massively parallel libraries of edited genomes based on established methods for sampling complex populations. We define specific attributes and metrics that are informative for describing a complex cell library and provide examples for estimating these values. We also connect this analysis to generic phenotyping approaches, using either pooled (typically via a selection assay) or isolate (often referred to as screening) phenotyping approaches. We approach this from the context of creating massively parallel, precisely edited libraries with one edit per cell, though the approach holds for other types of modifications, including libraries containing multiple edits per cell (combinatorial editing). This framework is a critical component for evaluating and comparing new technologies as well as understanding how a massively parallel edited cell library will perform in a given phenotyping approach.

genomics↗

bioRxiv2020

Role of nanoscale antigen organization on B-cell activation probed using DNA origami

Arraying vaccine immunogens in a multivalent form on the surface of virus-like particles is an important strategy used to enhance the efficacy of subunit vaccines. However, the impacts of antigen valency, spacing, and spatial organization on B cell triggering remain poorly understood. Here, we use DNA origami nanoparticles to create precise nanoscale organizations of a clinically-relevant HIV gp120 immunogen to systematically interrogate their impact on B cell triggering in vitro. We find that antigen dimers elicit monotonically increasing B cell receptor activation as inter-antigen spacing increases up to ~30 nm, and only 5 immunogens arrayed on the surface of a 3D particle are needed to elicit maximal B cell calcium signaling. Our results reveal design principles of viral and immunogen display that drive functional B cell responses.

immunology↗

bioRxiv2020

Arbitrary Boolean logical search operations on massive molecular file systems

DNA is an ultra-high-density storage medium that could meet exponentially growing worldwide demand for archival data storage if DNA synthesis costs declined sufficiently and random access of files within exabyte-to-yottabyte-scale DNA data pools were feasible. To overcome the second barrier, here we encapsulate data-encoding DNA file sequences within impervious silica capsules that are surface-labeled with single-stranded DNA barcodes. Barcodes are chosen to represent file metadata, enabling efficient and direct selection of sets of files with Boolean logic. We demonstrate random access of image files from an image database using fluorescence sorting with selection sensitivity of 1 in 106 files, which thereby enables 1 in 106N per N optical channels. Our strategy thereby offers retrieval of random file subsets from exabyte and larger-scale long-term DNA file storage databases, offering a scalable solution for random-access of archival files in massive molecular datasets.

bioengineering↗

bioRxiv2019

Bioproduction of single-stranded DNA from isogenic miniphage

Scalable production of gene-length single-stranded DNA (ssDNA) with sequence control has applications in homology directed repair templating, gene synthesis and sequencing, scaffolded DNA origami, and archival DNA memory storage. Biological production of circular single-stranded DNA (cssDNA) using bacteriophage M13 addresses these needs at low cost. A primary goal toward this end is to minimize the essential protein coding regions of the produced, exported sequence while maintaining its infectivity and production purity, with engineered regions of sequence control. Synthetic miniphage constitutes an ideal platform for bacterial production of isogenic cssDNA, using inserts of custom sequence and size to attain this goal, offering an inexpensive resource at milligram and higher synthesis scales. Here, we show that the Escherichia coli (E. coli) helper strain M13cp combined with a miniphage genome carrying only an f1 origin and a {beta}-lactamase-encoding (bla) antibiotic resistance gene enables the production of pure cssDNA with a minimum sequence genomic length of 1,676 nt directly from bacteria, without the need for additional purification from contaminating dsDNA, genomic DNA, or fragmented DNAs. Low-cost scalability of isogenic, custom-length cssDNA is also demonstrated for a sequence of 2,520 nt using a commercial bioreactor. We apply this system to generate cssDNA for the programmed self-assembly of wireframe DNA origami objects with exonuclease-resistant, custom-designed circular scaffolds that are purified with low endotoxin levels (<5 E.U./ml) for therapeutic applications. We also encode digital information that is stored on the genome with application to write-once, read-many archival data storage.

bioengineering↗

bioRxiv2017

Refactoring gene sequences for broad assembly standards compatibility

Four cloning standards in synthetic biology are BioBrick, BglBrick, MoClo and GoldenBraid, with each requiring their constitutive parts be compatible with the associated restriction enzymes. To standardize parts for the broadest usage, it would be useful to synthesize genes that are simultaneously compatible with all 4 popular assembly strategies. Here it is shown that using a defined set of rules, implemented in a computational program, any protein coding sequence can be made compatible with all four standards by silent mutations. Using a coding sequence as an input, all BioBrick, BglBrick, MoClo, and GoldenBraid restriction sites and chi recombination hot spots can be destroyed with silent mutations that approximate the codon usage of the organism. As an application, all open reading frames in the model organisms Escherichia Coli and Bacillus Subtilis are computationally refactored, showing the feasibility of implementing this umbrella strategy for synthesizing genes with the broadest compatibility.

synthetic biology↗

bioRxiv2017

Enzymatic synthesis of gene-length single-stranded DNA

Single-stranded DNA (ssDNA) increases the likelihood of homology directed repair with reduced cellular toxic ity, yet ssDNA synthesis strategies are limited by the maximum length attainable, as well control over nucleotide composition. Here, we apply purely enzymatic synthesis to generate ssDNA greater than 15 kb using asymmetric PCR, and illustrate the incorporation of diverse modified nucleotides for therapeutic and imaging applications.

bioengineering↗