Essay: Proposal: does quadruplex molecular beacon responsiveness and efficiency change when put into more complex environment

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  • Proposal: does quadruplex molecular beacon responsiveness and efficiency change when put into more complex environment
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Chapter 1 Introduction

1.1 Background

Importance of Cancer Screening

Among known diseases, cancer and heart disease are among the most recognizable and principle cause of mortality among those whom contracted them. Although cancer has led to fewer deaths than heart disease in the United States (at 22.0% compared to 23.4% of all deaths in 2015), an overall trend shows the mortality associated with heart disease has decreased largely since 1958. All the while, cancer associated mortality has remained constant despite current therapies. To put this in perspective, cancer is the second leading cause of death globally and approximately 70% of these associated deaths occur in middle- and low-income countries. Therefore development of effective therapies, through medical research and pharmaceuticals, has been a priority to reduce the mortality associated with cancer. Contributing to the ineffectiveness of cancer treatment is the lack of effective early detection methods and accurate chemotherapeutic treatments.
Today’s early detection methods have been observed to be very important for positive outcomes and increased survivability. When compared to late stage diagnoses, early stage diagnoses have been found to require less aggressive treatments since the tumor has not become metastatic and is still contained to one area.2, In order to effectively detect cancer early one must have access to screening and early diagnosis. The WHO describes screening as a way to identify abnormalities suggestive of a specific cancer within an individual who does not show symptoms yet. Early diagnosis focuses on the identification of early stage symptoms of specific cancers. Of the known cancers, only a few (breast, cervical, colorectal and oral cancer) have available screening methods. Of these specific cancer early detection methods, only a few can be reliably tested in middle- and low-income countries. This is due to the lack of imaging resources available, which are very costly techniques. To give some perspective, the regions where a majority of cancer deaths occur, low- or middle-income countries, have 3 to 21 times lower imaging resources available.2, Therefore development of early-detection methods of cancer that can be used worldwide are urgently required.

Importance of Nucleic Acids

The discovery of micro-RNA (miRNA, miR) in 1993 led to comprehensive studies that suggest that miRNA performs a crucial role in the early development of cancers. Micro-RNAs are defined as small RNAs that are between 19-28 nucleotides in length. Studies have shown that micro-RNA act as post-transcriptional silencing factors. As seen in figure 1.1, micro-RNA is incorporated into the RNA-induced silencing complex (RISC) that allows it to specifically bind to the 3’ untranslated region of messenger-RNA (mRNA) and cause degradation of the mRNA before it is translated into a functional protein. Recent studies have shown that specific families of miRNA are over- or under- expressed in tumor cells fairly consistently when compared to healthy tissue. These expressions patterns have led to miRNA possibly being used as biomarkers for the classification of specific cancers. These overexpressed miRNAs have even been found circulating in the blood stream during the early stages of tumor growth, which has led to them being used as biomarkers to distinguish cancer patients from healthy patients. It have been generally agreed upon that many studies have come to the conclusion that antisense oligonucleotides (ASOs) have shown the capability to bind with target miRNA and inhibit their activity. Thus, an oligonucleotide (ON) based sensor that is able to hybridize with target miRNA will lead to the inhibition of activity.

Many research groups have proposed ON-based sensor system with the use of different modifications. Research groups have used different ON modifiers, such as locked nucleic acids and phosphothioate backbones, in molecular beacons (MB) to enhance detection and specificity. They have also been observed to improve stability towards nuclease, a consistent issue with duplex molecular beacons. , A downside to this is the high cost of synthesizing molecular beacons with these modifiers. Other research groups have used gold nanoparticles to create molecular beacons able to sense for multiple miRNA of interest. These have been found to be effective and more specific as it can sense for the different miRNA that may be overexpressed in a specific cancer. Some of these systems have also included a conjugated aptamer to combine therapeutics and diagnostics. , Overall these systems have been extensively tested ex vivo but have been found to have low stability in vivo.

While the detection of miRNA has been shown to be an effective method for early detection of specific cancer, the furthest progressed system relies on laborious and expensive techniques such as gel-electrophoresis, RT-PCR and sequencing.7 This results in a system that is not available to the masses due to cost. Therefore, the development of an ON based sensor capable of producing a fluorescent single when hybridized to miRNA would have great potential as an inexpensive early detection system.

1.2 Design and Approach

Sensor Design

To develop a responsive ON-based sensor, the main requirement is the ability to convert between different conformations on a reversible basis. In addition, these conformations must all be stable under chosen conditions and able to reversible change in the presence of a target. Nucleic acids have been chosen as the scaffold for the reversible ON sensor because of the diversity of nucleic acid structures found in nature. These specific intramolecular folds (DNA duplex, intramolecular G-Quadruplex, cytosine quadruplex (i-motif), DNA triplex, ligand bound aptamer) can help with the reversible conformational change intended due to their increased stability for the folded structure. These switching ON sensors have been shown to work as stimuli-responsive systems, switching from a DNA duplex to a DNA hairpin, i-motif, G-Quadruplex, ligand bound aptamer, etc. , ,

To use ON-based sensor, a strand exchange mechanism known as toe-hold mediated exchange is employed. This mechanism uses a “toehold” overhang region that allows an additional longer complementary strand to hybridize and cause displacement of original partner strand to form a more stable duplex. This mechanism reveals that an oligonucleotide-based sensor is possible and can be made reversible.

To visualize and observe if the sensor is working and how efficiently it is working, Förster Resonance Energy Transfer (FRET) is utilized. FRET is the transfer of energy from a donor molecule to an acceptor molecule. This is seen through the overlap of the donor’s emission wavelengths and the acceptors absorbance wavelengths. FRET is especially useful for ON-based sensors because of its high dependence on distance between the donor and the acceptor. This distance dependence allows the observation of the sensor’s structural conformation. In addition, the acceptor in FRET can be either emissive (another fluorophore) or non-emissive, such as a quencher. This allows us to track different characteristics of the sensor and track the hybridization of the strands.

There are many factors that affect the stability of a ON-based sensor. When designing a sensor to be used in a blood test factors such as pH, ionic interactions and degradation due to nuclease should be taken into consideration. In understanding these environmental factors, it will help determine how they affect the sensor’s stabilization and efficiency. This will inform us as to how the sensor system will function in more complex environments such as: cell growth media, fetal bovine serum, live cells, etc.

Duplex Molecular Beacons

A well-studied and characterized approach to sensing for a specific ON sequence is through the use of molecular beacons. One of particular interest is the duplex molecular beacon. It has 2 strands in its system: guide strand (G) and sense strand (S). The sensing strand is a compliment to the miR target strand (T) and the guide strand is a shorter analogue of the target strand. The system is stable as a S:G duplex. When the miR target is present, it hybridizes to the toehold region and displaces the guide strand resulting in a S:T duplex. In order to visualize the sensor, a fluorophore modifier is added to the sense strand and a quencher modifier is added to the guide strand. When hybridized, in close proximity, the emission of the fluorophore is quenched by the quencher on the guide strand. When the S:T duplex is formed, the fluorophore is no longer being quench and there is a fluorescent emission.

While duplex molecular beacons are found to be functional ex vivo, they have been found to suffer from nuclease degradation in physiological conditions and low stability. There are backbone modifiers that have been added to increase stability of duplex sensors, but they do incur significant cost due to the added complexity of synthesis. Therefore, this study focuses on quadruplex structure as a scaffold for a molecular beacon.

Quadruplex Molecular Beacon

In contrast to duplex MB susceptibility to nuclease degradation, quadruplexes have been found to be more stable. The quadruplex structure has been found to be stable for extended periods of time in intracellular environments and in human blood. , this allows for a quadruplex MB to be used for stimuli responsiveness, while possessing nuclease resistance.

The quadruplex molecular beacon (QMB) used in this study incorporate both a G-quadruplex stem and long central recognition loop. The stem is made up of 3 Guanine-tetrads, which provide the structure and stability of the molecular beacon. The recognition loop is complimentary to the miR target. The fluorophore and quencher FRET pair are added to 5’ and 3’ end of the recognition loop. When the miR target is present, it hybridizes with the recognition loop of the QMB. There is then a conformational change from a closed intramolecular quadruplex to an open intermolecular duplex. This behavior describes a system that can be cycled between conformational states. When in its closed conformation, the fluorophore and quencher of the QMB are close enough to quench the fluorescent signal. When in its open conformation the fluorophore is no longer quenched, and a fluorescent signal is observed.

With separating the stem and recognition loop into structure and recognition, respectively, there is an increase in stability and specificity. Recent work has revealed that long recognition loops can be stable in a QMB with longer quadruplex stems.12 The ability to use longer recognition loops allows for a longer sequence to be sensed for. Together, these characteristics are very applicable to micro-RNA sensing.
Quadruplex structures found in nature have been characterized to have high stability, but QMB have not been extensively tested in vivo. Past studies have experimented with QMB ex vivo (in buffer), therefore there has not been much work into understanding their functionality and stability in more complex environments. Therefore, it would be beneficial to observe a QMB within more complex environment with multiple factors: ionic interactions, proteins, lipids, etc. The effects of these factors need to be understood in order to better optimize the system for potential in vivo or in vitro testing.

Investigation of QMB Responsiveness

Measuring Conformational Change of QMB

To determine relative stability of the QMB, thermal denaturation profiles will be made with particular attention paid to the melting temperature (Tm), where 50% of the QMB is denatured. To create a baseline to compare to, the QMB will first be tested in potassium phosphate buffer to simulate simple and isolated conditions. A thermal denaturation profile will not be done for the other various condition because other molecules and cofactors may alter the absorbance data.

To measure and observe the conformational change of QMB in environments of varying complexity, fluorescent spectroscopy will to employed. Fluorescent titration will be used to determine if there is a difference in sensor efficiency when comparing different environments. In addition, kinetic fluorescents studies will be conducted to determine the strand exchange kinetics. In both cases, FRET will be used to observe the conformational shift of the QMB.

Analysis

To interpret the raw spectroscopy data, Origin Pro software will be used. To determine thermal stability, the Boltzmann fit will be used to determine when 50% of the QMB is denatured. To determine strand exchange kinetics, the time dependent fluorescent spectroscopy experiments will be plotted and compared relative to each other. Efficiency of the QMB system will be analyzed through the peak area from the fluorescent titration experiments.

Objectives of Study

This study is conducted to understand if the quadruplex molecular beacon responsiveness and efficiency will change when put into more complex environments. Therefore, the folding of a QMB will be observed in a simple environment, potassium phosphate buffer. TO observe the potential effects of differing environments, the conformational change of a QMB will be observed in Dulbecco’s Modified Eagle media, Fetal Bovine Serum, and cancer cell lysate. The progress made in the optimization of quadruplex molecular beacon for micro-RNA targeting is described in this work.

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