WETLAB

Cortisol aptasensor formation and sensitivity

Formation using truncated aptamer and cDNA

Design

The truncated aptamer sequence was referred to us by Dr. Tarun Kumar Sharma from Gujarat Biotechnology University. This aptamer is a 14-mer, which is claimed to conform and bind to one of the prominent binding sites of the cortisol biomolecule. The cDNA (complementary ssDNA) was designed to complement the half length of the aptamer, making the cDNA 7bp long. Given below are the possible conformations of the 14-mer aptamer sequence obtained using DNA-fold software:

Test

To check if the truncated aptamer and cDNA were binding, a fluorescence-based test was performed using the flurolog equipment (the working of which is mentioned on the experiment page). The fluorescence-based tests were used to measure the optimum molar ratio between the aptamer and the cDNA and the incubation time required to form the aptasensor. To check the sensitivity of the truncated aptasensor and to determine the efficiency and working of the aptasensor, we performed plate reader experiments.

Build

The aptamer sequence was modified with a 5' fluorophore attachment, and the cDNA was modified with a quencher towards the 3' end. The fluorophore and quencher pair were decided based on their FRET efficiency. Our ideal choice for the fluorophore was fluorescein-based FAM with an excitation peak at 498 nm and an emission peak at 517 nm, and the quencher of choice was BHQ1, which has an absorption range of 480 to 580 nm with maximum absorption at 534 nm. The possible structure of the aptamer cDNA complex is given below:

Learn

We only observed a 10 per cent quenching from the above experimental data in 90 minutes. As shown above, the optimum molar ratio of 1:2 (trunc aptamer: cDNA) was used for the sensitivity experiment. The chosen aptasensor is not very sensitive within the range of 100 to 300 ng/ml cortisol, the physiological range in the human blood serum. Therefore, there are better quantifiers for cortisol biomolecules than the truncated aptasensor.

Formation of cortisol aptasensor using 85 mer aptamer and cDNA (44mer)

Learn

Due to the various challenges involved, and our need for knowledge about modifying the aptamer sequence to achieve effective turn-on, clickmer-based quantifiers were not a viable option for us.

Design

Aptamers are short, single-stranded DNA or RNA molecules that can bind to specific target molecules with high affinity and specificity. Aptamers can be modified with an alkyne group at one end to create a fluorescent aptasensor. This alkyne group can then be attached to an azide fluorescent dye using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction.
When the aptamer binds to its target biomolecule, it undergoes a conformational change that exposes the azide group attached to the fluorescent dye. The CuAAC reaction can then bind the fluorescent dye to the aptamer. This results in a fluorescent signal that can be detected using a fluorometer or microplate reader.
This method of creating a fluorescent aptasensor is called a "turn-on" mechanism because the fluorescence signal is only present when the aptamer is bound to its target biomolecule. This is in contrast to the "turn-off" mechanism, in which the fluorescence signal is detected when the aptamer is not attached to its target biomolecule.
Turn-on mechanisms are generally more desirable for aptasensors as they are more sensitive and less prone to false positives.

Build

We modified our selected aptamers by adding an azide group using Integrated DNA Technologies (IDT). To label the aptamers with the dye, we purchased Cy3-alkyne dye to perform a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction.

Test

We pitched this idea to multiple faculty members within our institute, and Dr. Reji Varghese made us aware of the possible difficulties, such as the efficiency of a click binding and also the difficulty in modifying a specific nucleotide within an aptamer sequence that is known to be attached to the binding site of the biomolecule.

Formation of cortisol aptasensor using 85 mer aptamer and cDNA designed based on kD value.

Learn

From the DBTL cycle, we learn that the ideal hybridisation time and molar ratios show similar trends with the cDNA 1 85-mer aptamer complex; hence, both cDNA 1 and cDNA 2 in combination with the 85-mer aptamer can be used to form the ideal cortisol aptasensor.

Design

Here, we used the same 85-mer aptamer mentioned in the previous iteration but changed the design of our cDNA binding to the aptamer that forms the aptasensor. This version of the cDNA was designed based on the kD value of the aptamer; the cDNA pair compared to the kD value of the cortisol binding with the 85-mer aptamer. As shown below, multiple complementary sequences were designed to find a sequence with a higher kD value than -16.1 that would not affect the binding and formation of the FRET system designed. The chosen cDNA had a kD value of -10.3, and even though there were multiple possible structures, the conformations didn't affect the binding position of the cDNA, hence the interaction of the fluorophore quencher pair.

Build

Test

To check if the 85-mer aptamer and the designed cDNA were binding, a fluorescence-based test was performed using the flurolog equipment (working is mentioned in the experiment page” link”) to confirm binding between the aptamer and cDNA. We also conducted an isothermal titration calorimetric experiment to obtain the kD and delta g value; the fluorescence-based tests were used to measure the ideal molar ratio between the aptamer and the cDNA and the incubation time required for the formation of the aptasensor. To check the sensitivity of the obtained aptasensor, we performed plate reader experiments to determine the efficacy and detection limit of the aptasensor.

Serotonin aptasensor formation and sensitivity

Iteration 1 Iteration 2

44-mer serotonin aptamer and 20-mer cDNA

Learn

The results above show that the hybridization of the cDNA and aptamer causes an increase in fluorescence. In contrast, the interaction between the fluorophore quencher pair should cause a decrease in fluorescence. This could be because the partial binding of the cDNA occurred elsewhere within the aptamer, causing a change in the conformation of the aptamer, thereby exposing the fluorophore molecule. This theory would explain the increase in fluorescence over time observed after the hybridization of the cDNA with the aptamer.

Design

The 44-mer aptamer was derived from the literature obtained from [r]; the possible conformations of the serotonin aptamer are given below. The aptamer had a kd value of -30 nm and a delta g value of -10.62 kcal/mol. This version of the cDNA was designed based on the kD value of the aptamer; the cDNA pair compared to the kD value of the serotonin binding with the 44-mer aptamer. As shown below, multiple complementary sequences were designed to find a sequence with a higher kD value than -30.1 that would not affect the binding and formation of the designed FRET system. The chosen cDNA had a kD value of -9.9 kcal/mol, and the possible conformation didn’t affect the binding position of the cDNA, hence the interaction of the fluorophore quencher pair.

Build

The aptamer sequence was modified with a 5' fluorophore attachment, and the cDNA was modified with a quencher towards the 3' end. The fluorophore and quencher pair were decided on their FRET efficiency; our ideal choice of the fluorophore was FAM, which is a fluorescein-based fluorophore with an excitation peak at 498 nm and an emission peak at 517 nm, and the quencher of choice was BHQ1 which has an absorption range of BHQ1 is from 480 to 580 nm with maximum absorption at 534 nm. These sequences were then ordered from IDT for further testing. Attached below is the possible hybridised duplex structure acquired using the duplex fold software Attached below are the possible hybridised duplex structures acquired using the duplex fold software: Link
https://rna.urmc.rochester.edu/RNAstructureWeb/Servers/DuplexFold/DuplexFold.html

Test

To check if the 44-mer serotonin aptamer and the designed cDNA were binding, a fluorescence-based test was performed using the Teacan plate reader equipment (working is mentioned in the experiment page” link”) to confirm binding between the aptamer and cDNA. We also conducted an isothermal titration calorimetric experiment to obtain the kD and delta g value; the fluorescence-based tests were used to measure the ideal molar ratio between the aptamer and the cDNA and the incubation time required for the formation of the aptasensor. To check the sensitivity of the obtained aptasensor, we performed plate reader experiments to determine the efficacy and detection limit of the aptasensor.

44-mer serotonin aptamer and 20-mer cDNA

Learn

The fluorescence-based test conducted using the plate reader gives us an observable quenching of 30% on the formation of the aptasensor using this cDNA, as seen with the cortisol aptasensor, which is a drastic improvement from the previously noticed increase in fluorescence upon hybridisation of the cDNA (12-mer) and the aptamer. Sensitivity tests also show increased fluorescence intensity upon binding with our Serotonin biomolecule. However, no more remarkable perceivable fold change is observed compared to the previously formed aptasensor.

Design

Here, we used the same 44-mer aptamer mentioned in the previous iteration but changed the design of our cDNA binding to the aptamer that forms the aptasensor. Our team also designed a cDNA based on the inputs received from Dr. Tarun Kumar Sharma from Gujarat Biotechnology University, who suggested we design the cDNA to complement at least half the length of the aptamer, so this version of our cDNA 1 was 20 bp long with delta g value - (-29.6 kcal/mol).

Build

Test

DRYLAB DBTL CYCLES

Iteration 1 Iteration 2 Iteration 3

Learn

Due to the various challenges involved, and our lack of knowledge about how to modify the aptamer sequence to achieve effective turn-on, clickmer-based quantifiers are not a viable option at this time.

Design

Aptamers are short, single-stranded DNA or RNA molecules that can bind to specific target molecules with high affinity and specificity. To create a fluorescent aptasensor, aptamers can be modified with an alkyne group at one end. This alkyne group can then be attached to an azide fluorescent dye using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction.

When the aptamer binds to its target biomolecule, it undergoes a conformational change that exposes the azide group on the fluorescent dye. The CuAAC reaction can then occur, attaching the fluorescent dye to the aptamer. This results in a fluorescent signal that can be detected using a fluorometer or microplate reader.

This method of creating a fluorescent aptasensor is called a "turn-on" mechanism because the fluorescence signal is only present when the aptamer is bound to its target biomolecule. This is in contrast to a "turn-off" mechanism, in which the fluorescence signal is present when the aptamer is not bound to its target biomolecule.

Turn-on mechanisms are generally more desirable for aptasensors because they are more sensitive and less prone to false positives.

Build

We used Integrated DNA Technologies (IDT) modifications to add an azide group to our selected aptamers. We planned to purchase Cy3-alkyne dye and perform a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction to label the aptamers with the dye. https://rna.urmc.rochester.edu/RNAstructureWeb/Servers/DuplexFold/DuplexFold.html

Test

We pitched this idea to multiple faculty members within our institute and Dr. Reji Varghese made us aware of the possible difficulties such as the efficiency of a click binding and also the difficulty in modifying a certain nucleotide within an aptamer sequence that is known to be attached to the binding site of the biomolecule.

Learn

We believe that the aptasensor method is a more practical choice for quantifying cortisol than clickmer-based methods, based on the following two factors:

Abundance of literature: There is a much larger body of literature available on aptasensors than on clickmer-based methods for cortisol quantification. This means that there are more well-established protocols and procedures available for aptasensor-based methods, and that there is a larger community of researchers who are familiar with and experienced in using these methods.

Fewer theoretical problems and questions: Aptasensors are based on a well-established principle (FRET), and there are few theoretical problems or questions associated with their use. Clickmer-based methods, on the other hand, are a newer technology, and there are still some theoretical problems and questions that need to be addressed.

Design

This mechanism works on the principle of Fluorescence resonance energy transfer (FRET) is a distance-dependent non-radiative transfer of energy from one fluorescent molecule (the donor) to another fluorescent molecule (the acceptor). It occurs when the two molecules are within close proximity (typically 10 nm or less) and their emission and excitation spectra overlap. We attach a fluorescent molecule (fluorophore) to the 5' end of the aptamer sequence and a quencher molecule to the 3' end of the complementary strand. When the cDNA and the aptamer bind to form a partial duplex structure, the fluorophore and the quencher molecule come close together, which quenches the fluorescence. This is our aptasensor complex. When the aptasensor complex interacts with the target biomolecule (cortisol), the cDNA strand is displaced. The free aptamer strand then changes conformation, allowing it to bind to the biomolecule. This causes an increase in fluorescence, which is quantified to measure the level of cortisol present. In other words, the aptasensor complex is like a switch. When the cDNA strand is bound to the aptamer, the switch is off. When the biomolecule binds to the aptamer, the switch is turned on. The fluorescence signal is directly proportional to the amount of biomolecule present, so we can use the fluorescence intensity to measure the biomolecule concentration.

Build

The aptamer sequence was modified with a 5' fluorophore attachment, and the cDNA was modified with a quencher towards the 3’ end. The fluorophore and quencher pair were decided on their FRET efficiency; our ideal choice of the fluorophore was FAM, which is a fluorescein-based fluorophore with an excitation peak at 498 nm and an emission peak at 517 nm, and the quencher of choice was BHQ1 which has an absorption range of BHQ1 is from 480 to 580 nm with maximum absorption at 534 nm. These sequences were then ordered from IDT for further testing.

Test

Various tests were performed for various types and concentration of aptasensors these test can be viewed in the results and proof of concept page.

Learn

We realised why toehold switched would not be the ideal quantification method for miRNAs

It will have to include translatory machinery, which requires specific enzymes.

An amplification step would be required to bring the concentration of miRNA to detectable levels, increasing the cost and complexity of the chip.

Storage of RNA-based quantifiers would require specialised conditions.

Enzymes used for translation must be freeze-dried and stored in the microchip.

Leaky expression would still be present because of low levels of enzyme activity.

During this stage, we were doubtful about using Toehold switches. This is when we came across nanoprobes, which were DNA-based, did not require enzymes and had very good detection limits.[3]

Design

Toehold switches are synthetic RNAs that mimic messenger RNAs. They contain a recognition sequence for a specific stimulus in the form of a specific “input” RNA and a recognition sequence that the protein-synthesizing machinery (ribosome) needs to bind to initiate the translation of a fused protein-coding sequence into its encoded protein product. In the absence of the “input” RNA, the toehold switch is kept in its OFF state by forming a hairpin structure that uses part of the “input” recognition sequence and the ribosome recognition sequence, which is kept inaccessible. The toehold switch is turned on when a stimulating “input” RNA binds to the toehold and induces the hairpin structure to open up, giving the ribosome access to its recognition sequence to start the synthesis of the encoded protein downstream [1] Using a toehold switch, we intended to quantify our chosen miRNAs- miR124 and miR132. This could be made possible by tagging a GFP protein gene as the reporter sequence at the 3’ end of the toehold switch, which will get translated only in the presence of the trigger miRNA. Hence, the sample will show fluorescence in the presence of the respective miRNA, which could then be quantified to measure the amount of miRNAs in the sample.

Build

We were using the switchMI designer tool developed by the 2021 iGEM team UParis BME and NUPACK software to build the sequence of our toehold switches. [2]

Superfolded GFP was chosen as the reporter sequence.

We were able to generate a preliminary set of sequences based on this.

Test

To test the feasibility of our idea and protocols, we talked to Tania, a PhD from IISER PUNE.

To test the feasibility of our idea and protocols, we talked to Tania, a PhD from IISER PUNE. To test the feasibility of our idea and protocols, we talked to Tania, a PhD from IISER PUNE. To test the feasibility of our idea and protocols, we talked to Tania, a PhD from IISER PUNE. To test the feasibility of our idea and protocols, we talked to Tania, a PhD from IISER PUNE.

HARDWARE DBTL CYCLES

Iteration 1 Iteration 2 Iteration 3 Iteration 4 Iteration 5 Iteration 6

Learn

The led was able to detect higher intensities well but could not respond to lower levels of light. We would need a more sensitive unconventional but cheap sensor.

Design

A container containing a source to mimic fluorescence (green which is expected from biological samples), a sensor to detect it and a self-contained microcontroller module for data collection and display

Build

Box out of Cardboard, green led used as a source, used a photodiode for its high sensitivity to green light and fast response times and Arduino Uno as the microcontroller

Test

Setup the container in a dark room with Arduino powered and outputting data to a laptop. Varied the voltage across the led in several small steps to achieve lowest possible levels of light via direct control from Arduino

Learn

The phototransistor achieved saturation current production at moderate intensities of light but was more sensitive to lower levels of light compared to the photodiode. Signal was also amplified by using a large resistance (300kohm) in series with phototransistor. However, the detected emission was still visible to naked eye. We would need to make instrument more sensitive or amplify further.

Design

Build

Box out of Cardboard, green led used as a source, used a phototransistor for its higher sensitivity to lower light and an Arduino Uno as the microcontroller

Test

Learn

Photodarlington was much more sensitive to low levels of light. The sensor still picked up the light even after 1 million ohms ins series with led. However, reading was unreliable because of large fluctuation in signal. This was arising from the fact that Arduino uses Pulse Width Modulated signal to control led voltage and brightness, leading to rapid flickering. Increasing resistance (over 300kohm) in series with Darlington system did not reliably amplify signal anymore.

Design

Similar design as earlier. Led was placed directly above the photodarlington ( sensor is minimally sensitive to light coming from +/- 5 degrees from directly vertically above it).

Build

Box out of Cardboard, green led used as a source, used a photodarlington (in series with 300kohm resistor for amplification) for the highest sensitivity to lower light, and an Arduino Uno as the microcontroller.

Test

Similar to above. 1 million ohms was placed in series with led to lower intensity of light as much as possible.

Learn

The signal detected by the transistor was steady and showed good behavior. Detected amounts of light demonstrated nearly lower than naked eye detection. Detection was now limited by accuracy of ADC reading on Arduino, which is 10 microvolts. Laptops and devices were turned away or switched off as device was sensitive to even such minute light. Device was now ready to be tested with non-electronic signals.

Design

Similar design as earlier. Led was placed directly above the photodarlington ( sensor is minimally sensitive to light coming from +/- 5 degrees from directly vertically above it).

Build

Test

Similar to above. 1 million ohms was placed in series with led to lower intensity of light as much as possible.

Learn

Device performed exceptionally well. Reliably picked up readings until 4th order of dilution (10^-4 of stock concentration). 4th order and 5th order dilution were distinct; however, they were not reliably separated enough. Great care was needed for 3rd of dilution and below. Laptops were turned away from system and set at minimum brightness. All devices were moved off table and no physical contact with table was needed for most accurate readings. Any further noise signals were averaged out using an algorithm which gave effective readings every 20 seconds. To further improve sensitivity or accuracy, a voltage regulated DC source along with 16-bit external ADC (Arduinos is 10-bit) might be required. Now we pray biological samples are bright enough

Design

Custom Led at 490nm (blue) driven by DC source (in series with 100 kohm resistor to avoid photobleaching) was used to excite PET chemical sample to mimic biological fluorescence, used a photodarlington (in series with 300 kohm resistor for amplification) as sensor, an Arduino Uno for data collection and display.

Build

Interior of box was painted black to absorb any remnant blue light to reduce noise. Sensor was placed at 90 degrees and away from blue light source (sensor is minimally sensitive to light coming from +/- 5 degrees from directly vertically above it). Furthermore, A plane separated the blue light from the sensor to minimize undesirable signals. Cuvettes (or whatever they are called) were used to hold samples and were placed directly above sensor.

Test

Learn

All biological samples showed great distinction between stock and biomarker added scenarios. Detected fluorescence was close to or above 3rd order dilution readings from PET, making the setup less fragile to change while reliable reading data. Addition of nanoprobes further improved readings and gave better signals. Signals were stable. Development of ultra-sensitive, cheap fluorimeter, tailored o detect biomarker signals of green fluorescence was successfully developed.

Design

Custom Led at 490nm (blue) driven by DC source (in series with 100 kohm resistor to avoid photobleaching) was used to excite wetlab biological samples, used a photodarlington (in series with 300 kohm resistor for amplification) as sensor, an Arduino Uno for data collection and display.

Build

Test

Biological samples at realistic concentrations and volumes were used. Readings for cases of without biomarker and with biomarker for all aptamers, mirna132 and mirna124 was done

References

Tolentino, J. C., & Schmidt, S. L. (2018). DSM-5 Criteria and Depression Severity: Implications for Clinical Practice. Frontiers in psychiatry, 9, 450. doi.org/10.3389/fpsyt.2018.00450 https://doi.org/10.3389/fpsyt.2018.00450 (1 December 2011)- World Health Organisation.
https://www.who.int/news-room/fact-sheets/detail/depression