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 open-source toolkit features a user-friendly web tool, interactive graphics, up to 100 times 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.

Programming cell-free biosensors with DNA strand displacement circuits.

Cell-free biosensors are powerful platforms for monitoring human and environmental health. Here, we expand their capabilities by interfacing them with toehold-mediated strand displacement circuits, a dynamic DNA nanotechnology that enables molecular computation through programmable interactions between nucleic acid strands. We develop design rules for interfacing a small molecule sensing platform called ROSALIND with toehold-mediated strand displacement to construct hybrid RNA-DNA circuits that allow fine-tuning of reaction kinetics. We use these design rules to build 12 different circuits that implement a range of logic functions (NOT, OR, AND, IMPLY, NOR, NIMPLY, NAND). Finally, we demonstrate a circuit that acts like an analog-to-digital converter to create a series of binary outputs that encode the concentration range of the molecule being detected. We believe this work establishes a pathway to create 'smart' diagnostics that use molecular computations to enhance the speed and utility of biosensors.

ROSALIND: Rapid Detection of Chemical Contaminants with In Vitro Transcription Factor-Based Biosensors.

ROSALIND (RNA Output Sensors Activated by Ligand Induction) is an in vitro biosensing system that detects small molecules using regulated transcription reactions. It consists of three key components: (1) RNA polymerases, (2) allosteric protein transcription factors, and (3) synthetic DNA transcription templates that together regulate the synthesis of a fluorescence-activating RNA aptamer. The system can detect a wide range of chemicals including antibiotics, small molecules, and metal ions. We have demonstrated that ROSALIND can be lyophilized and transported at ambient conditions for water testing on-site. Here, we describe how to set up a ROSALIND reaction for detecting various chemical contaminants in water using a model transcription factor as well as how to build a new ROSALIND sensor.

2'-fluoro-modified pyrimidines enhance affinity of RNA oligonucleotides to HIV-1 reverse transcriptase.

Nucleic acid aptamers can be chemically modified to enhance function, but modifying previously selected aptamers can have nontrivial structural and functional consequences. We present a reselection strategy to evaluate the impact of several modifications on preexisting aptamer pools. RNA aptamer libraries with affinity to HIV-1 reverse transcriptase (RT) were retranscribed with 2'-F, 2'-OMe, or 2'-NH2 pyrimidines and subjected to three additional selection cycles. RT inhibition was observed for representative aptamers from several structural families identified by high-throughput sequencing when transcribed with their corresponding modifications. Thus, reselection identified specialized subsets of aptamers that tolerated chemical modifications from unmodified preenriched libraries. Inhibition was the strongest with the 2'-F-pyrimidine (2'-FY) RNAs, as compared to inhibition by the 2'-OMeY and 2'-NH2Y RNAs. Unexpectedly, a diverse panel of retroviral RTs were strongly inhibited by all 2'-FY-modified transcripts, including sequences that do not inhibit those RTs as unmodified RNA. The magnitude of promiscuous RT inhibition was proportional to mole fraction 2'-FY in the transcript. RT binding affinity by 2'-FY transcripts was more sensitive to salt concentration than binding by unmodified transcripts, indicating that interaction with retroviral RTs is more ionic in character for 2'-FY RNA than for unmodified 2'-OH RNA. These surprising features of 2'-FY-modified RNA may have general implications for applied aptamer technologies.

Cell-free biosensors for rapid detection of water contaminants.

Lack of access to safe drinking water is a global problem, and methods to reliably and easily detect contaminants could be transformative. We report the development of a cell-free in vitro transcription system that uses RNA Output Sensors Activated by Ligand Induction (ROSALIND) to detect contaminants in water. A combination of highly processive RNA polymerases, allosteric protein transcription factors and synthetic DNA transcription templates regulates the synthesis of a fluorescence-activating RNA aptamer. The presence of a target contaminant induces the transcription of the aptamer, and a fluorescent signal is produced. We apply ROSALIND to detect a range of water contaminants, including antibiotics, small molecules and metals. We also show that adding RNA circuitry can invert responses, reduce crosstalk and improve sensitivity without protein engineering. The ROSALIND system can be freeze-dried for easy storage and distribution, and we apply it in the field to test municipal water supplies, demonstrating its potential use for monitoring water quality.

Design and Optimization of a Cell-Free Atrazine Biosensor.

Recent advances in cell-free synthetic biology have spurred the development of in vitro molecular diagnostics that serve as effective alternatives to whole-cell biosensors. However, cell-free sensors for detecting manmade organic water contaminants such as pesticides are sparse, partially because few characterized natural biological sensors can directly detect such pollutants. Here, we present a platform for the cell-free detection of one critical water contaminant, atrazine, by combining a previously characterized cyanuric acid biosensor with a reconstituted atrazine-to-cyanuric acid metabolic pathway composed of several protein-enriched bacterial extracts mixed in a one pot reaction. Our cell-free sensor detects atrazine within an hour of incubation at an activation ratio superior to previously reported whole-cell atrazine sensors. We also show that the response characteristics of the atrazine sensor can be tuned by manipulating the ratios of enriched extracts in the cell-free reaction mixture. Our approach of utilizing multiple metabolic steps, encoded in protein-enriched cell-free extracts, to convert a target of interest into a molecule that can be sensed by a transcription factor is modular. Our work thus serves as an effective proof-of-concept for a scheme of "metabolic biosensing", which should enable rapid, field-deployable detection of complex organic water contaminants.

A Primer on Emerging Field-Deployable Synthetic Biology Tools for Global Water Quality Monitoring.

Tracking progress towards Target 6.1 of the United Nations Sustainable Development Goals, "achieving universal and equitable access to safe and affordable drinking water for all", necessitates the development of simple, inexpensive tools to monitor water quality. The rapidly growing field of synthetic biology has the potential to address this need by taking DNA-encoded sensing elements from nature and reassembling them to create field-deployable 'biosensors' that can detect pathogenic or chemical water contaminants. Here we describe water quality monitoring strategies enabled by synthetic biology and compare them to previous approaches used to detect three priority water contaminants: fecal pathogens, arsenic, and fluoride in order to explain the potential for engineered biosensors to simplify and decentralize water quality monitoring. We also briefly discuss expanding biosensors to detect emerging contaminants including metals and pharmaceuticals. We conclude with an outlook on the future of biosensor development, in which we discuss adaptability to emerging contaminants, outline current limitations, and propose steps to overcome the field's outstanding challenges to facilitate global water quality monitoring.

Design of a Transcriptional Biosensor for the Portable, On-Demand Detection of Cyanuric Acid.

Rapid molecular biosensing is an emerging application area for synthetic biology. Here, we engineer a portable biosensor for cyanuric acid (CYA), an analyte of interest for human and environmental health, using a LysR-type transcription regulator (LTTR) from Pseudomonas within the context of Escherichia coli gene expression machinery. To overcome cross-host portability challenges of LTTRs, we rationally engineered hybrid Pseudomonas-E. coli promoters by integrating DNA elements required for transcriptional activity and ligand-dependent regulation from both hosts, which enabled E. coli to function as a whole-cell biosensor for CYA. To alleviate challenges of whole-cell biosensing, we adapted these promoter designs to function within a freeze-dried E. coli cell-free system to sense CYA. This portable, on-demand system robustly detects CYA within an hour from laboratory and real-world samples and works with both fluorescent and colorimetric reporters. This work elucidates general principles to facilitate the engineering of a wider array of LTTR-based environmental sensors.

Poly-Target Selection Identifies Broad-Spectrum RNA Aptamers.

Aptamer selections often yield distinct subpopulations, each with unique phenotypes that can be leveraged for specialized applications. Although most selections aim to attain ever higher specificity, we sought to identify aptamers that recognize increasingly divergent primate lentiviral reverse transcriptases (RTs). We hypothesized that aptamer subpopulations in libraries pre-enriched against a single RT may exhibit broad-spectrum binding and inhibition, and we devised a multiplexed poly-target selection to elicit those phenotypes against a panel of primate lentiviral RTs. High-throughput sequencing and coenrichment/codepletion analysis of parallel and duplicate selection trajectories rapidly narrowed the list of candidate aptamers by orders of magnitude and identified dozens of priority candidates for further screening. Biochemical characterization validated a novel aptamer motif and several rare and unobserved variants of previously known motifs that inhibited recombinant RTs to varying degrees. These broad-spectrum aptamers also suppressed replication of viral constructs carrying phylogenetically diverse RTs. The poly-target selection and coenrichment/codepletion approach described herein is a generalizable strategy for identifying cross-reactivity among related targets from combinatorial libraries.

Modular cell-internalizing aptamer nanostructure enables targeted delivery of large functional RNAs in cancer cell lines.

Large RNAs and ribonucleoprotein complexes have powerful therapeutic potential, but effective cell-targeted delivery tools are limited. Aptamers that internalize into target cells can deliver siRNAs (<15 kDa, 19-21 nt/strand). We demonstrate a modular nanostructure for cellular delivery of large, functional RNA payloads (50-80 kDa, 175-250 nt) by aptamers that recognize multiple human B cell cancer lines and transferrin receptor-expressing cells. Fluorogenic RNA reporter payloads enable accelerated testing of platform designs and rapid evaluation of assembly and internalization. Modularity is demonstrated by swapping in different targeting and payload aptamers. Both modules internalize into leukemic B cell lines and remained colocalized within endosomes. Fluorescence from internalized RNA persists for ≥2 h, suggesting a sizable window for aptamer payloads to exert influence upon targeted cells. This demonstration of aptamer-mediated, cell-internalizing delivery of large RNAs with retention of functional structure raises the possibility of manipulating endosomes and cells by delivering large aptamers and regulatory RNAs.

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.

In Vivo Analysis of Infectivity, Fusogenicity, and Incorporation of a Mutagenic Viral Glycoprotein Library Reveals Determinants for Virus Incorporation.

UNLABELLED: Enveloped viruses utilize transmembrane surface glycoproteins to gain entry into target cells. Glycoproteins from diverse viral families can be incorporated into nonnative viral particles in a process termed pseudotyping; however, the molecular mechanisms governing acquisition of these glycoproteins are poorly understood. For murine leukemia virus envelope (MLV Env) glycoprotein, incorporation into foreign viral particles has been shown to be an active process, but it does not appear to be caused by direct interactions among viral proteins. In this study, we coupled in vivo selection systems with Illumina next-generation sequencing (NGS) to test hundreds of thousands of MLV Env mutants for the ability to be enriched in viral particles and to perform other glycoprotein functions. NGS analyses on a subset of these mutants predicted that the residues important for incorporation are in the membrane-proximal external region (MPER), particularly W127 and W137, and the residues in the membrane-spanning domain (MSD) and also immediately flanking it (T140 to L163). These predictions were validated by directly measuring the impact of mutations in these regions on fusogenicity, infectivity, and incorporation. We suggest that these two regions dictate pseudotyping through interactions with specific lipid environments formed during viral assembly. IMPORTANCE: Researchers from numerous fields routinely exploit the ability to manipulate viral tropism by swapping viral surface proteins. However, this process, termed pseudotyping, is poorly understood at the molecular level. For murine leukemia virus envelope (MLV Env) glycoprotein, incorporation into foreign viral particles is an active process, but it does not appear to occur through direct viral protein-protein interactions. In this study, we tested hundreds of thousands of MLV Env mutants for the ability to be enriched in viral particles as well as perform other glycoprotein functions. Our analyses on a subset of these mutants predict that the glycoprotein regions embedded in and immediately flanking the viral membrane dictate active incorporation into viral particles. We suggest that pseudotyping occurs through specific lipid-protein interactions at the viral assembly site.

FASTAptamer: A Bioinformatic Toolkit for High-throughput Sequence Analysis of Combinatorial Selections.

High-throughput sequence (HTS) analysis of combinatorial selection populations accelerates lead discovery and optimization and offers dynamic insight into selection processes. An underlying principle is that selection enriches high-fitness sequences as a fraction of the population, whereas low-fitness sequences are depleted. HTS analysis readily provides the requisite numerical information by tracking the evolutionary trajectory of individual sequences in response to selection pressures. Unlike genomic data, for which a number of software solutions exist, user-friendly tools are not readily available for the combinatorial selections field, leading many users to create custom software. FASTAptamer was designed to address the sequence-level analysis needs of the field. The open source FASTAptamer toolkit counts, normalizes and ranks read counts in a FASTQ file, compares populations for sequence distribution, generates clusters of sequence families, calculates fold-enrichment of sequences throughout the course of a selection and searches for degenerate sequence motifs. While originally designed for aptamer selections, FASTAptamer can be applied to any selection strategy that can utilize next-generation DNA sequencing, such as ribozyme or deoxyribozyme selections, in vivo mutagenesis and various surface display technologies (peptide, antibody fragment, mRNA, etc.). FASTAptamer software, sample data and a user's guide are available for download at http://burkelab.missouri.edu/fastaptamer.html.