I am passionate about improving human health and quality of life using functional nucleic acids. Specifically, my past, current & future research aims to improve the selection and characterization of small molecule-binding aptamers and ribozymes towards the ultimate goal of developing both in vitro and in vivo biosensors for high-throughput drug screening and disease diagnosis.
Honors & Awards
NSERC Postdoctoral Fellowship, The Natural Sciences and Engineering Research Council of Canada (NSERC) (2013-2015)
Bachelor of Science, Carleton University (2007)
Doctor of Philosophy, Carleton University (2012)
Christina Smolke, Postdoctoral Faculty Sponsor
Opportunities in the design and application of RNA for gene expression control.
Nucleic acids research
2016; 44 (7): 2987-99
The past decade of synthetic biology research has witnessed numerous advances in the development of tools and frameworks for the design and characterization of biological systems. Researchers have focused on the use of RNA for gene expression control due to its versatility in sensing molecular ligands and the relative ease by which RNA can be modeled and designed compared to proteins. We review the recent progress in the field with respect to RNA-based genetic devices that are controlled through small molecule and protein interactions. We discuss new approaches for generating and characterizing these devices and their underlying components. We also highlight immediate challenges, future directions and recent applications of synthetic RNA devices in engineered biological systems.
View details for DOI 10.1093/nar/gkw151
View details for PubMedID 26969733
Screening and identification of DNA aptamers to tyramine using in vitro selection and high-throughput sequencing.
ACS combinatorial science
Aptamers are synthetic single-stranded DNA or RNA sequences that can fold into tertiary structures allowing them to interact with and bind to targets with high affinity and specificity. This paper describes the first selection and identification of DNA aptamers able to recognize the biogenic amine tyramine. To successfully isolate aptamers to this challenging small molecule target, the SELEX methodology was adapted by combining a systematic strategy to increase the selection stringency and monitor enrichment success. As the benefits of applying high-throughput sequencing (HTS) in SELEX experiments is becoming more clear, this method was employed in combination with bioinformatics analysis to evaluate the utility of the selection strategy and to uncover new potential high affinity sequences. Based on the presence of consensus regions (sequence families) and family similarities (clusters), 15 putative aptamers to tyramine were identified. A recently described workflow approach to perform a primary screening and characterization of the aptamer candidates by micro-equilibrium dialysis and by microscale thermophoresis was next leveraged. These candidate aptamers exhibited dissociation constant (Kd) values in the range of 0.2-152 µM with aptamer Tyr_10 as the most promising one followed by aptamer Tyr_14. These aptamers could be used as promising molecular recognition tools for the development of inexpensive, robust and innovative biosensor platforms for the detection of tyramine in food and beverages.
View details for DOI 10.1021/acscombsci.5b00163
View details for PubMedID 27057927
Analysis of In Vitro Aptamer Selection Parameters
JOURNAL OF MOLECULAR EVOLUTION
2015; 81 (5-6): 150-161
Nucleic acid aptamers are novel molecular recognition tools that offer many advantages compared to their antibody and peptide-based counterparts. However, challenges associated with in vitro selection, characterization, and validation have limited their wide-spread use in the fields of diagnostics and therapeutics. Here, we extracted detailed information about aptamer selection experiments housed in the Aptamer Base, spanning over two decades, to perform the first parameter analysis of conditions used to identify and isolate aptamers de novo. We used information from 492 published SELEX experiments and studied the relationships between the nucleic acid library, target choice, selection methods, experimental conditions, and the affinity of the resulting aptamer candidates. Our findings highlight that the choice of target and selection template made the largest and most significant impact on the success of a de novo aptamer selection. Our results further emphasize the need for improved documentation and more thorough experimentation of SELEX criteria to determine their correlation with SELEX success.
View details for DOI 10.1007/s00239-015-9708-6
View details for Web of Science ID 000365713700005
In Vitro Screening and in Silico Modeling of RNA-Based Gene Expression Control
ACS CHEMICAL BIOLOGY
2015; 10 (11): 2463-2467
Molecular tools for controlling gene expression are essential for manipulating biological systems. One class of tools includes RNA switches that incorporate RNA-based sensors, known as aptamers. However, most switches reported to date are responsive to toxic molecules or to endogenous metabolites. For effective conditional control, switches must incorporate RNA aptamers that exhibit selectivity against such endogenous metabolites. We report a systematic approach which combines a rapid in vitro assay and an in silico model to support an efficient, streamlined application of aptamers into RNA switches. Model predictions were validated in vivo and demonstrate that the RNA switches enable selective and programmable gene regulation. We demonstrate the method using aptamers that bind the FDA-approved small molecule (6R)-folinic acid, providing access to new molecular targets for gene expression control and much-needed clinically relevant tools for advancing RNA-based therapeutics.
View details for DOI 10.1021/acschembio.5b00518
View details for Web of Science ID 000365462700006
Comprehensive Analytical Comparison of Strategies Used for Small Molecule Aptamer Evaluation
2015; 87 (17): 8608-8612
Nucleic acid aptamers are versatile molecular recognition agents that bind to their targets with high selectivity and affinity. The past few years have seen a dramatic increase in aptamer development and interest for diagnostic and therapeutic applications. As the applications for aptamers expand, the need for a more standardized, stringent, and informative characterization and validation methodology increases. Here we performed a comprehensive analysis of a panel of conventional affinity binding assays using a suite of aptamers for the small molecule target ochratoxin A (OTA). Our results highlight inconsistency between conventional affinity assays and the need for multiple characterization strategies. To mitigate some of the challenges revealed in our head-to-head comparison of aptamer binding assays, we further developed and evaluated a set of novel strategies that facilitate efficient screening and characterization of aptamers in solution. Finally, we provide a workflow that permits rapid and robust screening, characterization, and functional verification of aptamers thus improving their development and integration into novel applications.
View details for DOI 10.1021/acs.analchem.5b02102
View details for Web of Science ID 000360773100003
View details for PubMedID 26192270
An in solution assay for interrogation of affinity and rational minimer design for small molecule-binding aptamers.
Aptamers are short single-stranded oligonucleotides that fold into unique three-dimensional structures, facilitating selective and high affinity binding to their cognate targets. It is not well understood how aptamer-target interactions affect regions of structure in an aptamer, particularly for small molecule targets where binding is often not accompanied by a dramatic change in structure. The DNase I footprinting assay is a classical molecular biology technique for studying DNA-protein interactions. The simplest application of this method permits identification of protein binding where DNase I digestion is inhibited. Here, we describe a novel variation of the classical DNase I assay to study aptamer-small molecule interactions. Given that DNase I preferentially cleaves duplex DNA over single-stranded DNA, we are able to identify regions of aptamer structure that are affected by small molecule target binding. Importantly, our method allows us to quantify these subtle effects, providing an in solution measurement of aptamer-target affinity. We applied this method to study aptamers that bind to the mycotoxin fumonisin B1, allowing the first identification of high affinity putative minimers for this important food contaminant. We confirmed the binding affinity of these minimers using a magnetic bead binding assay.
View details for DOI 10.1039/c5an01075f
View details for PubMedID 26336657
Selection and Characterization of a Novel DNA Aptamer for Label-Free Fluorescence Biosensing of Ochratoxin A
2014; 6 (8): 2435-2452
Nucleic acid aptamers are emerging as useful molecular recognition tools for food safety monitoring. However, practical and technical challenges limit the number and diversity of available aptamer probes that can be incorporated into novel sensing schemes. This work describes the selection of novel DNA aptamers that bind to the important food contaminant ochratoxin A (OTA). Following 15 rounds of in vitro selection, sequences were analyzed for OTA binding. Two of the isolated aptamers demonstrated high affinity binding and selectivity to this mycotoxin compared to similar food adulterants. These sequences, as well as a truncated aptamer (minimal sequence required for binding), were incorporated into a SYBR® Green I fluorescence-based OTA biosensing scheme. This label-free detection platform is capable of rapid, selective, and sensitive OTA quantification with a limit of detection of 9 nM and linear quantification up to 100 nM.
View details for DOI 10.3390/toxins6082435
View details for Web of Science ID 000341427200015
View details for PubMedID 25153252
Kinetic and equilibrium binding characterization of aptamers to small molecules using a label-free, sensitive, and scalable platform.
2014; 86 (7): 3273-3278
Nucleic acid aptamers function as versatile sensing and targeting agents for analytical, diagnostic, therapeutic, and gene-regulatory applications, but their limited characterization and functional validation have hindered their broader implementation. We report the development of a surface plasmon resonance-based platform for rapid characterization of kinetic and equilibrium binding properties of aptamers to small molecules. Our system is label-free and scalable and enables analysis of different aptamer-target pairs and binding conditions with the same platform. This method demonstrates improved sensitivity, flexibility, and stability compared to other aptamer characterization methods. We validated our assay against previously reported aptamer affinity and kinetic measurements and further characterized a diverse panel of 12 small molecule-binding RNA and DNA aptamers. We report the first kinetic characterization for six of these aptamers and affinity characterization of two others. This work is the first example of direct comparison of in vitro selected and natural aptamers using consistent characterization conditions, thus providing insight into the influence of environmental conditions on aptamer binding kinetics and affinities, indicating different possible regulatory strategies used by natural aptamers, and identifying potential in vitro selection strategies to improve resulting binding affinities.
View details for DOI 10.1021/ac5001527
View details for PubMedID 24548121
Facile characterization of aptamer kinetic and equilibrium binding properties using surface plasmon resonance.
Methods in enzymology
2014; 549: 451-466
Nucleic acid aptamers find widespread use as targeting and sensing agents in nature and biotechnology. Their ability to bind an extensive range of molecular targets, including small molecules, proteins, and ions, with high affinity and specificity enables their use in diverse diagnostic, therapeutic, imaging, and gene-regulatory applications. Here, we describe methods for characterizing aptamer kinetic and equilibrium binding properties using a surface plasmon resonance-based platform. This aptamer characterization platform is broadly useful for studying aptamer-ligand interactions, comparing aptamer properties, screening functional aptamers during in vitro selection processes, and prototyping aptamers for integration into nucleic acid devices.
View details for DOI 10.1016/B978-0-12-801122-5.00019-2
View details for PubMedID 25432760
- Development of a DNA aptamer for direct and selective homocysteine detection in human serum RSC ADVANCES 2013; 3 (46): 24415-24422
Aptamer base: a collaborative knowledge base to describe aptamers and SELEX experiments
DATABASE-THE JOURNAL OF BIOLOGICAL DATABASES AND CURATION
Over the past several decades, rapid developments in both molecular and information technology have collectively increased our ability to understand molecular recognition. One emerging area of interest in molecular recognition research includes the isolation of aptamers. Aptamers are single-stranded nucleic acid or amino acid polymers that recognize and bind to targets with high affinity and selectivity. While research has focused on collecting aptamers and their interactions, most of the information regarding experimental methods remains in the unstructured and textual format of peer reviewed publications. To address this, we present the Aptamer Base, a database that provides detailed, structured information about the experimental conditions under which aptamers were selected and their binding affinity quantified. The open collaborative nature of the Aptamer Base provides the community with a unique resource that can be updated and curated in a decentralized manner, thereby accommodating the ever evolving field of aptamer research. DATABASE URL: http://aptamer.freebase.com.
View details for DOI 10.1093/database/bas006
View details for Web of Science ID 000304920200004
View details for PubMedID 22434840
Challenges and opportunities for small molecule aptamer development.
Journal of nucleic acids
2012; 2012: 748913-?
Aptamers are single-stranded oligonucleotides that bind to targets with high affinity and selectivity. Their use as molecular recognition elements has emerged as a viable approach for biosensing, diagnostics, and therapeutics. Despite this potential, relatively few aptamers exist that bind to small molecules. Small molecules are important targets for investigation due to their diverse biological functions as well as their clinical and commercial uses. Novel, effective molecular recognition probes for these compounds are therefore of great interest. This paper will highlight the technical challenges of aptamer development for small molecule targets, as well as the opportunities that exist for their application in biosensing and chemical biology.
View details for DOI 10.1155/2012/748913
View details for PubMedID 23150810
Screening and Initial Binding Assessment of Fumonisin B-1 Aptamers
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
2010; 11 (12): 4864-4881
Fumonisins are mycotoxins produced by Fusarium verticillioides and F. proliferatum, fungi that are ubiquitous in corn (maize). Insect damage and some other environmental conditions result in the accumulation of fumonisins in corn-based products worldwide. Current methods of fumonisin detection rely on the use of immunoaffinity columns and high-performance liquid chromatography (HPLC). The use of aptamers offers a good alternative to the use of antibodies in fumonisin cleanup and detection due to lower costs and improved stability. Aptamers are single-stranded oligonucleotides that are selected using Systematic Evolution of Ligands by EXponential enrichment (SELEX) for their ability to bind to targets with high affinity and specificity. Sequences obtained after 18 rounds of SELEX were screened for their ability to bind to fumonisin B(1). Six unique sequences were obtained, each showing improved binding to fumonisin B(1) compared to controls. Sequence FB(1) 39 binds to fumonisin with a dissociation constant of 100 ± 30 nM and shows potential for use in fumonisin biosensors and solid phase extraction columns.
View details for DOI 10.3390/ijms11124864
View details for Web of Science ID 000285708000006
View details for PubMedID 21614178
Computational approaches toward the design of pools for the in vitro selection of complex aptamers
RNA-A PUBLICATION OF THE RNA SOCIETY
2010; 16 (11): 2252-2262
It is well known that using random RNA/DNA sequences for SELEX experiments will generally yield low-complexity structures. Early experimental results suggest that having a structurally diverse library, which, for instance, includes high-order junctions, may prove useful in finding new functional motifs. Here, we develop two computational methods to generate sequences that exhibit higher structural complexity and can be used to increase the overall structural diversity of initial pools for in vitro selection experiments. Random Filtering selectively increases the number of five-way junctions in RNA/DNA pools, and Genetic Filtering designs RNA/DNA pools to a specified structure distribution, whether uniform or otherwise. We show that using our computationally designed DNA pool greatly improves access to highly complex sequence structures for SELEX experiments (without losing our ability to select for common one-way and two-way junction sequences).
View details for DOI 10.1261/rna.2102210
View details for Web of Science ID 000283047900020
View details for PubMedID 20870801