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.
Full text
Available for:
EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
Full text
Available for:
FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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.
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'-NH
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'-NH
Y 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.
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.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
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.
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.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
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.
Display omitted
Combinatorial selections strategies are tools to design sequences with desirable properties. However, existing tools that analyze these data require significant computational expertise. The corresponding author and colleagues designed the webtool FASTAptameR 2.0 to minimize this technical barrier and allow researchers to still answer nuanced questions about their sequence populations.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
PDF
