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Bioinformatics, Computational and Systems Biology

Bioinformatics, Computational and Systems Biology - Poster Session D

Poster A8 - Optimizing Toehold Switches for the Detection of Cervical Precancer microRNAs

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Co-Author(s)

Renee A. Jaranson

Undergraduate Bioengineering Student at the University of Maryland, College Park
Optical Therapeutics & Nanotechnology Laboratory, University of Maryland, College Park
Elkridge, Maryland, United States
Navya Bansal (they/them/theirs)

Undergraduate Researcher
University of Maryland International Genetically Engineered Machine, United States
Mia Jocic (she/her/hers)

Undergraduate Researcher
University of Maryland International Genetically Engineered Machine, United States
JM

Jonathon Meyer, N/A (They/He)

Undergraduate Researcher
University of Maryland International Genetically Engineered Machine
Westminster, Maryland, United States
Lauren Applegate

Undergraduate Researcher
University of Maryland International Genetically Engineered Machine, United States
JW

Jonathan Wu

Undergraduate Researcher
University of Maryland International Genetically Engineered Machine, United States
Abhiraman Senthilkumar (he/him/his)

Undergraduate Researcher
University of Maryland International Genetically Engineered Machine
Frederick, Maryland, United States
Aditri Gadigi (she/her/hers)

Undergraduate Researcher
University of Maryland International Genetically Engineered Machine
Monmouth Junction, New Jersey, United States

Presenting Author(s)

Kevin Zhang (he/him/his)

Undergraduate Researcher
University of Maryland International Genetically Engineered Machine
Ijamsville, Maryland, United States

Co-Author(s)

Krista Adu-Osei (she/her/hers)

Undergraduate Researcher
University of Maryland International Genetically Engineered Machine, United States
Rebecca Lu (she/her/hers)

Undergraduate Researcher
University of Maryland International Genetically Engineered Machine, United States
Graham M. Ferguson (he/him/his)

Undergraduate Researcher
University of Maryland International Genetically Engineered Machine
College Park, Maryland, United States
HH

Haider Hussain

Undergraduate Researcher
University of Maryland International Genetically Engineered Machine, United States
HL

Hannah LaPadula

Undergraduate Researcher
University of Maryland International Genetically Engineered Machine, United States
Sarah Firdaus (she/her/hers)

Undergraduate Researcher
University of Maryland International Genetically Engineered Machine
Rockville, Maryland, United States

Introduction: In 2022, lower- and middle-income countries (LMICs) bore 94% of total cervical cancer deaths, highlighting the global disparity in treating the fourth-most common cancer in women. Access to diagnostic tests such as pap smears and VIA screening have decreased cervical cancer mortality rates in higher income countries. These tests can be hard to access in LMICs due to issues with cost, discomfort, reliable refrigeration, and reliance on specialized equipment or technicians. As a result, many patients in LMICs do not receive adequate cervical cancer screening.

A promising method for addressing these issues involves targeting an emerging class of biomarkers called microRNAs (miRNAs), which can be found in easily accessible bodily fluids such as urine. Literature suggests that three upregulated miRNA targets in the urine of cervical precancer patients (miR-21-5p, miR-155-5p, and miR-199a-5p) may offer a high level of specificity and sensitivity in detecting cervical precancer.

To quantify these miRNAs, a system involving synthetic RNA devices called toehold switches is proposed. Toehold switches are riboregulators that express a downstream protein in the presence of specific RNA biomarkers. While toehold switches have been optimized for RNA and mRNA detection, they have not been tested in vitro with smaller RNA targets such as miRNAs.

Thus far, three toehold switches have been designed, modeled, and tested in vivo with E.coli. Two out of three toeholds show specific miRNA detection and are currently being tested in vitro for future translation onto a paper-based screening device.

Materials and

Methods: Three toehold switches were designed based on 2014 Green et al. specifications with a trigger sequence (complementary to the miRNA biomarkers), a complementary stem region, a linker sequence, and a red fluorescent protein (RFP) coding region. Toehold secondary structures were confirmed using RNAstructure and toehold-miRNA binding affinities were predicted to be favorable by RNAup. Toehold and miRNA gBlocks were ordered from IDT with vector overlap segments at the ends to facilitate insertion into chloramphenicol and ampicillin resistance plasmids respectively via gibson assembly. Corresponding toehold and miRNA plasmids were transformed into BL21(DE3) E.coli cells and plated with chloramphenicol and ampicillin to select cells with both plasmids. BL21 cells were also transformed with miRNA plasmids that do not correspond with the toehold plasmid to check for nonspecific toehold expression. Successful colonies were then transferred to liquid culture where plasmid expression was induced with IPTG before incubating for eight hours at 37C. Sample RFP was identified via visual inspection after pelleting and washing cells with water.

Results, Conclusions, and Discussions: The secondary structures generated by RNAstructure for each toehold switch confirmed that the toehold sequences had a high probability of folding to form toehold conformations. RNAstructure also confirmed that these structures were favorable, with low free energies of formation. The RNAstructure bifolds of the miRNA-toehold complexes showed binding between the miRNAs and toeholds at the trigger region and favorable free energies of formation. These estimations were expanded upon by the RNAup server, which predicted miRNA-toehold dissociation constants in the pico and femtomolar range, or essentially one to one binding.

BL21 E.coli cells transformed with corresponding 21-5p and 199a-5p toehold and miRNA plasmids produced RFP as observed upon visual inspection. Surprisingly, the 155-5p toehold and miRNA plasmids did not appear to produce any RFP. None of the toeholds visibly expressed RFP in E.coli containing non-corresponding miRNA plasmids. Based on these findings, it can be concluded that two out of the three toeholds can produce RFP upon miRNA binding and that the toeholds bind specifically to their target sequences. To confirm these results quantitatively, RFP within BL21 pellets will be measured via fluorescence with a sulforhodamine standard curve.

In order to translate these toeholds onto a cell-free paper-based device, further testing is required to characterize and optimize these toeholds in vitro and on paper. In the next few months, the binding affinity of the toeholds will be confirmed using fluorescence resonance energy transfer and PAGE gel methods. The limit of detection for the miRNAs will be determined in vitro using a transcription/translation kit that enables RFP production from the toehold and miRNA plasmids outside of cells. Toeholds will be modified (changing the length of the trigger region, making substitutions in the stem and trigger regions) to test the effect on RFP production. For translation into a screening device, the RFP reporter gene could be replaced with nanoluciferase for cheaper quantification. Finally, these reactions will be transferred onto paper.

Barriers in accessing screening tests contribute significantly to the high cervical cancer mortality rates seen in LMICs. Paper-based assays employing RNA devices such as toehold switches could improve access to cervical cancer screening.

Acknowledgements (Optional):