Project Description

A Riboswitch is a special sequence within mRNA comprised of two essential segments: the aptamer region and the expression region. In the aptamer region, specific small molecules or proteins of interest bind with high affinity^[6]. This means the aptamers are highly selective, ensuring they exclusively bind to the desired molecules. When this binding occurs, the expression region undergoes a conformational change. This change can either unveil or conceal the ribosome binding site (RBS), consequently permitting or obstructing the translation of nucleotides downstream. In our experimentation, we have targeted the permitting of transcription/translation. Riboswitches are invaluable tools, often harnessed as detection methods. The utilization of riboswitches lies in the ability to alter the sequence surrounding the RBS. This customization allows for the creation of a measurable reporter.

In our project, we've engineered specific riboswitches designed to respond to target biomarkers. These riboswitches were specifically designed so that when exposed to the intended biomarkers, they undergo a conformational change. The resulting change transcribes an enzyme used in our detection process.

We used the Salis Lab’s Riboswitch Calculator to design our riboswitch sequences. The Riboswitch Calculator is a physics-based model that uses thermodynamics to alter the pre- and post-aptamer sequences that optimize activation ratio, essentially changing certain nucleotides to provide detectable ON and OFF states for regulating translation. Researchers have successfully created cell-free genetically encoded biosensors capable of detecting small molecules and nucleic acids. An automated platform was designed to transform protein-binding RNA aptamers into riboswitch sensors. These sensors can function effectively in cell-free assays, the ideology the Penn State team utilized. The Salis team engineered 35 riboswitches capable of sensing proteins, including human monomeric C-reactive protein, human interleukin-32γ, and phage MS2 coat protein. These riboswitch sensors adjusted output expression levels by up to 16-fold in response to protein concentrations found in human serum. Through computational analysis, the researchers identified two distinct mechanisms governing riboswitch-mediated regulation of translation rates. By refining the protein-binding aptamer regions, they improved the design accuracy of the sensors. This advancement broadens the toolkit for cell-free sensors and demonstrates the application of computational design in creating protein-sensing riboswitches, holding potential for future use in affordable medical diagnostics^[7]. The second paper introduced initially to the team by Dr. Howard Salis used riboswitches to regulate gene expression. Riboswitches are specialized RNA molecules that change shape in response to chemicals, thereby regulating gene expression. This mechanism links detection to cellular response directly. However, understanding how their genetic sequence influences riboswitch switching and activation, especially when altering the ligand-binding aptamer region, has remained unclear. The Salis Lab has developed a statistical thermodynamic model that predicts the relationship between sequence, structure, and function for riboswitches that control gene expression. This model was validated both inside cells and in cell-free transcription-translation assays. Using this model, they performed automated computational design to create 62 synthetic riboswitches. These riboswitches utilized six different RNA aptamers to detect various chemicals (theophylline, tetramethylrosamine, fluoride, dopamine, thyroxine, 2,4-dinitrotoluene) and triggered gene expression up to 383-fold. The model explains how the aptamer's structure, affinity to the chemical, energy required for switching, and cellular crowding work together to control riboswitch activation. The model-based approach not only sheds light on the physical mechanisms underlying ligand-induced RNA shape changes but also enables the creation of cell-free and bacterial sensors for a wide array of applications^[8]. De Novo DNA, a Salis Lab spin-off, holds the Riboswitch Calculator. This model implements the expertise above allowing for researchers to design specific riboswitches.

As a means to reduce diagnostic timing the team introduces SensoREX, an innovative in vitro biomarker measurement tool. SensoREX leverages Riboswitch-Enabled eXpression (REX) technology to provide precise and reliable biomarker detection. Comprising a plasmid encoding the riboswitch, a cell-free system, and engineered trehalose, SensoREX integrates these with a standard glucometer. The operational procedure is simple: a blood sample from a finger prick is added to SensoREX. The user initiates the process by obtaining an initial glucose reading, followed by a specified incubation period. After this interval, a final glucose reading is taken. These readings are then input into the Biomarker Calculator, which translates the data into the corresponding biomarker concentration. The concentration variations relate to the stage of the disease and/or treatment progress. SensoREX stands out due to its swiftness, affordability, and user-friendly nature, making biomarker measurement widely accessible. The inherent high affinities and specificities of riboswitches render SensoREX well-suited for diagnostic applications and continuous disease monitoring. The standardized system allows for future biomarker testing with indicators other than the ones selected by the team.

As we began our project with minimal riboswitch expertise, our team was guided by Dr. Salis to select potential aptamers consisting of an average of seventy base pairs. The larger the aptamer size, the more complicated the riboswitch and conformational changes would become. The team combed through hundreds of potential options to identify six aptamers with human implications. The six selected were Monomeric C-Reactive Protein (mCRP), Interleukin-32γ, Thyroxine, Basic Fibroblast Growth Factor (bFGF), Vascular Endothelial Growth factor (VEGF), and Bovine Thrombin.


Monomeric C-Reactive Protein

C-reactive protein (CRP) is a protein that significantly rises during infections or inflammation. It is produced in the form of native CRP (nCRP) which can break down into monomeric CRP (mCRP) at sites of inflammation^[9]. Initially used as an infection and cardiovascular marker, recent studies show CRP's role in inflammation, complement pathway, apoptosis, phagocytosis, and other biological processes. Differentiating between CRP isoforms (nCRP and mCRP) is crucial, but commercial antibodies for mCRP are scarce, limiting research. NCRP exhibits anti-inflammatory activities, activating complement pathways and promoting apoptosis. MCRP, in contrast, attracts immune cells to inflammation sites and delays cell death. The mCRP isoform in local inflammation and infection sites was the primary focus of the two.

In our development of SensoREX we discovered limitations and challenges to using biomarkers in disease applications^[1].

Developing biomarkers presents several challenges: