Biology subjects

Burke, D. H.

Publications and source records attributed to Burke, D. H..

5 recordsLinked to original sources

bioRxiv2023

Differentiation SELEX approach identifies RNA aptamers with different specificities for HIV-1 capsid assembly forms

The HIV-1 capsid protein (CA) assumes distinct assembly forms during replication, each presenting unique, solvent-accessible surfaces that facilitate multifaceted functions and host factor interactions. However, contributions of individual CA assemblies remain unclear, as the evaluation of CA in cells presents several technical challenges. To address this need, we sought to identify CA assembly form-specific aptamers. Aptamer subsets with different specificities emerged from within a highly converged, pre-enriched aptamer library previously selected to bind the CA hexamer lattice. Subsets were either highly specific for CA lattice or bound both CA lattice and CA hexamer. We further evaluated four representatives to reveal aptamer structural features required for binding, highlighting interesting features and challenges in aptamer structure determination. Importantly, our aptamers bind biologically relevant forms of CA and we demonstrate aptamer-mediated affinity purification of CA from cell lysates without virus or host modification. Thus, we have identified CA assembly form-specific aptamers that represent exciting new tools for the study of CA.

molecular biology↗

bioRxiv2022

FASTAptameR 2.0: A Web Tool for Combinatorial Sequence Selections

Combinatorial selections are powerful strategies for identifying biopolymers with specific biological, biomedical, or chemical characteristics. Unfortunately, most available software tools for high-throughput sequencing analysis have high entrance barriers for many users because they require extensive programming expertise. FASTAptameR 2.0 is an R-based reimplementation of FASTAptamer designed to minimize this barrier while maintaining the ability to answer complex sequence-level and population-level questions. This opensource toolkit features a user-friendly web tool, interactive graphics, up to 100x faster clustering, an expanded module set, and an extensive user guide. FASTAptameR 2.0 accepts diverse input polymer types and can be applied to any sequence-encoded selection.

bioinformatics↗

bioRxiv2020

Lipidated poly(amino acid) nanostructures as versatile therapeutic delivery vehicles

Poly(amino acid)s are a diverse and capable class of polymers with significant potential for utilization in a wide variety of drug delivery applications. A sub-class of these biomaterials known as lipidated poly(amino acid)s (LPAAs) are amphiphiles composed of both hydrophobic and hydrophilic domains yielding interesting physical properties. In this article, we describe our efforts in developing a novel class of lysine and valine containing LPAAs synthesized via hexadecylamine initiated N-carboxyanhydride ring-opening polymerization (NCA-ROP). These highly hydrophobic LPAAs were found capable of undergoing hydrophobically-driven self-assembly into small nanostructures as well as being forced into larger nanostructures using a novel dump-and-stir nanoprecipitation process. This process yielded fine control over resulting nanoparticle size and cargo entrapment. Furthermore, cell-targeting DNA aptamer modification of doxorubicin-loaded LPAA nanoparticles induced significant death of co-incubated Non-Hodgkin Lymphoma cells providing exciting evidence of the therapeutic potential of this novel biomaterials-based delivery device.

bioengineering↗

bioRxiv2018

Poly-Target Selection Identifies RNA Broad-Spectrum Inhibitors of HIV Reverse Transcriptases

Aptamer selections often yield distinct subpopulations, each with unique phenotypes that can be leveraged for specialized applications. RNA aptamers that bind HIV-1 reverse transcriptase (RT) exhibit potent RT inhibition and suppress viral replication when targeting the strain-specific RT that they were originally selected to bind, but some of these same aptamers fail against single-point mutant and phylogenetically-diverse RTs. We hypothesized that a subset of the total aptamer population in libraries pre-enriched against a single RT may exhibit broad-spectrum RT binding and inhibition, and we devised a multiplexed Poly-Target selection approach to elicit those phenotypes against a panel of diverse primate lentiviral RTs. High-throughput sequencing of starting, negative, and final libraries, followed by analysis of coenrichment and codepletion in parallel and duplicate selection trajectories, narrowed the list of candidate aptamers by orders of magnitude. Biochemical characterization of candidates identified a novel aptamer motif and several rare and unobserved variants of previously-known motifs that inhibited recombinant RTs from HIV-1, HIV-2 and SIV to varying degrees. These broad-spectrum aptamers also suppressed replication of viral constructs carrying phylogenetically-diverse RTs. The Poly-Target selection and coenrichment approach described herein is a generalizable strategy for identifying broad-spectrum behavior and cross-reactivity among related targets from combinatorial libraries.

molecular biology↗

bioRxiv2017

A Fluorescent Split Aptamer for Visualizing RNA-RNA Assembly In Vivo

RNA-RNA assembly governs key biological processes and is a powerful tool for engineering synthetic genetic circuits. Characterizing RNA assembly in living cells often involves monitoring fluorescent reporter proteins, which are at best indirect measures of underlying RNA-RNA hybridization events and are subject to additional temporal and load constraints associated with translation and activation of reporter proteins. In contrast, RNA aptamers that sequester small molecule dyes and activate their fluorescence are increasingly utilized in genetically-encoded strategies to report on RNA-level events. Split-aptamer systems have been rationally designed to generate signal upon hybridization of two or more discrete RNA transcripts, but none directly function when expressed in vivo. We reasoned that the improved physiological properties of the Broccoli aptamer enable construction of a split-aptamer system that could function in living cells. Here we present the Split-Broccoli system, in which self-assembly is nucleated by a thermostable, three-way junction RNA architecture and fluorescence activation requires both strands. Functional assembly of the system approximately follows second order kinetics in vitro and improves when cotranscribed, rather than when assembled from purified components. Split-Broccoli fluorescence is digital in vivo and retains functional modularity when fused to RNAs that regulate circuit function through RNA-RNA hybridization, as demonstrated with an RNA Toehold switch. Split-Broccoli represents the first functional split-aptamer system to operate in vivo. It offers a genetically-encoded and nondestructive platform to monitor and exploit RNA-RNA hybridization, whether as an all-RNA, stand-alone AND gate or as a tool for monitoring assembly of RNA-RNA hybrids.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC=\"FIGDIR/small/109306_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (18K):\norg.highwire.dtl.DTLVardef@9d07d1org.highwire.dtl.DTLVardef@16c6a41org.highwire.dtl.DTLVardef@b3e6a6org.highwire.dtl.DTLVardef@eba2cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗