iGEM 2023 — Team hs-china

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Laboratory notebooks are essential tools for scientists, researchers, and students alike. They are used to record observations, methodologies, results, and interpretations during experiments. Team HS-China refined their experimental design step by step using 136 pages of lab notes and achieved relatively good experimental results.

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## Overview

Design-Build-Test-Learn is an iterative process used to systematically optimize and refine our biological systems
for the detection of COPD.

Design: In this phase, we designed a biological system based on the desired function or performance.
Build: After the design is completed, we used molecular biology techniques to construct the designed biological
parts and systems.

Test: After the construction is completed, these biological systems would be tested under experimental conditions
to determine whether their function and performance meet the design expectations.
Learn: Based on the data obtained in the testing phase, we evaluated and modified the design to further improve in
the next iteration.

This cyclical process is critical in our project because it allows us to quickly and systematically optimize
biological systems until the desired function or performance is achieved.

## Design

COPD (Chronic Obstructive Pulmonary Disease) is a lung disease that impacts around 212.3 million people around the
globe, with the majority of cases occurring in developing countries such as China, where there is a significant lack
of hospitals/clinics in its vast underdeveloped rural areas. In addition, conventional methods for the diagnosis of
COPD have a multitude of problems. The most notable problem is that current diagnosis methods are not COPD-specific.
Vital capacity, for example, is also used to diagnose pneumonia. Conventional methods are rough, non-specific, and
heavily dependent on experiences of the diagnosing doctor. Therefore, what we are aiming for is an efficient,
specific, and entirely objective method for COPD diagnosis.

## Build

MicroRNA is central to our project. We found some specific miRNAs whose concentration is associated with the
pathological progression of COPD. miRNA-1274a, for example, affects apoptosis and cellular senescence. That’s why
the concentration of miRNA-1274a in lung tissues is correlated to COPD (Gon et al., 2019).

## Test

We used RT-qPCR, a combination of RT-PCR and qPCR, for testing miR-1274a and miR-223 performances in an in vitro
reaction under various concentrations. After analysis of our results, we excluded miR-1274a as our target of
detection due to its poor performance with RT-qPCR, the ‘golden standard’ and conventional miRNA detection method.
MiR-223 yielded significantly better results than miR-1274a and negative control U6 (Figure 1), therefore we
selected it as our primary target of detection. RT-qPCR results would be compared to our MB-ERC2 system results in
the future.

![](https://static.igem.wiki/teams/4664/wiki/content/engineering/img01.png)

*Figure 1 100 pM U6, miR-1274a, and miR-223*

## Learn and redesign

RT-qPCR, while being the ‘golden standard’ for miRNA detection, still has several short-comings, the most notable
one being efficiency. The entire procedure of RT-qPCR is several hours in length, and involves multiple steps
requiring different reaction conditions (i.e. temperature). Furthermore, human errors during transitions between the
steps may lead to misdiagnosis. Therefore, we are aiming for a convenient (e.g. isothermal and one-step only) and
efficient method for miRNA detection.

## Build

Rolling circle amplification (RCA) is a method commonly used in exponential amplification of target sequences,
while cas12a cleavage of fluorophore-quencher reporters could generate fluorescence signals that can be potentially
quantified. Therefore, we decided to combine the two, drawing inspiration from He et al. (2023a). Before we
attempted a one-step, isothermal reaction system, we first tried tandem combinations of ligation, RCA and Cas12a.
Our first test trial was a three step assay (ligation-RCA-Cas12), lasting approximately 5-6 hours. After that, we
attempted a two step method (with ligation and RCA jointly carried out instead of separately, as in the three step
assay), which shortened the reaction time to 3-4 hours. Then, after both intermediary assays yielded satisfactory
results, we began testing a one-step reaction (Figure 2).

![](https://static.igem.wiki/teams/4664/wiki/content/engineering/img02.jpg)

*Figure 2 One-step, isothermal reaction system*

## Test

Our one-pot isothermal MB-ERC2 system yielded results comparable to those from RT-qPCR. Furthermore, reaction time
ranges from 20 minutes minimum to 2 hours maximum, significantly shorter than both RT-qPCR and the intermediary
assays.

More information: <https://2023.igem.wiki/hs-china/results>

## Build

After we achieved initial success, we began a series of experiments intended to determine the optimal concentrations
of reagents used in the MB-ERC2 system (Figure 3 as an example for Phi29 concentration optimization，more information:
https://2023.igem.wiki/hs-china/results). Furthermore, we designed a series of padlocks to observe the effects of
crRNA recognition zone location and miRNA binding zone mismatches on reaction kinetics. Through these experiments, we
determined the optimal conditions for the MB-ERC2 system.

![](https://static.igem.wiki/teams/4664/wiki/content/engineering/img03.png)

*Figure 3 Final fluorescence levels for Phi29 concentration optimization*

## References

Gon, Y., Shimizu, T., Mizumura, K., Maruoka, S., & Hikichi, M. (2019). Molecular techniques for respiratory diseases:
MicroRNA and extracellular vesicles. Respirology, 25(2), 149–160. <https://doi.org/10.1111/resp.13756>

He, Y., Wen, Y., Tian, Z., Hart, N. T., Han, S., Hughes, S. J., & Zeng, Y. (2023a). A one-pot isothermal Cas12-based
assay for the sensitive detection of microRNAs. Nature Biomedical Engineering.
<https://doi.org/10.1038/s41551-023-01033-1>

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## Overview

---

Our wetlab activities could be divided into two parts: RT-qPCR and our MB-ERC2 (miRNA biomarker-based exponential
RCA Cas12a/CRISPR) system. The RT-qPCR trials were performed as comparison groups for the EXTRA-CRISPR groups.
Moreover, through examining the results yielded by RT-qPCR, the golden standard for miRNA detection, we selected
miR-223, which gave significantly better performance, as our final target biomarker. The Cas12a-based assay trials
demonstrated the efficiency and accuracy of our detection method. Through single-variable experiments, we optimised
the reaction system and verified that our standard reaction system has the best reaction kinetics.

## RT-qPCR

---

RT-qPCR (Reverse Transcription Quantitative Real-time Polymerase Chain Reaction), combining RT-PCR and qPCR,
involves the detection and quantification of RNA. The procedure involves the enzyme reverse transcriptase converting
total RNA or messenger RNA (mRNA) to complementary DNA (cDNA). This cDNA is then amplified and used in quantitative
PCR (qPCR) or real-time PCR to detect specific targets. This PCR uses a number of fluorescent chemicals to quantify
the amount of DNA at each cycle in real-time.
Moreover, the RT-qPCR method has become an essential tool of detecting MicroRNA (miRNA), which is the key indicator
for detecting the presence of COPD.
Our first RT-qPCR trial starts with the miRNA biomarkers: miR-223 and miR-1274a. They are extracted from a fridge,
then undergo centrifugation for 3000r 3min, and dilute with water, and pre-heated in an incubator. Then 4 PCR tubes
are labelled miR-223 and miR-1274a. The next step is to add mRQ buffer and mRQ enzymes as well as miRNA samples to
respective PCR tubes, all operating on ice. The tunes are then transferred to the incubator for body temperature, an
hour. Lastly, they are taken out and placed in a drying oven, with addition of DEPC-treated water, and then stored
in the fridge for later usage.

![](https://static.igem.wiki/teams/4664/wiki/experiment/experiment/experiment/screen-shot-2023-10-09-at-10-09-02-pm.png)

After preparing cDNA, RT-qPCR could then be prepared by 8 PCR tubes (4 each for miR-223 and miR-1274a), with mRQ
buffer each tube, miRNA sample respectively and mRQ enzyme each tube. All the tubes are then controlled at 37ºC, 1 h
in an incubator. Finally, they are sent to a driving oven, with addition of DEPC-treated water.

### RCA

---

#### Padlock Design

The ends of the padlock are the miRNA binding sites. They are complementary to our target miRNAs (miR-223 and
miR-1274a). The influence of mismatches was investigated with padlocks M1-14 in the Padlock Probe Optimization
section below.
A cas12a detection zone is also located on the padlock. The detection module contains sequences complementary to
the crRNA used in the synthesis of RNP. An optimization of the location of the detection modules was performed
with padlocks 2-4 in the Padlock Probe Optimization section.

![](https://static.igem.wiki/teams/4664/wiki/experiment/experiment/experiment/screen-shot-2023-10-09-at-10-11-35-pm.png)

#### SplintR Ligation

While the conventional T4 ligase also ligases DNA-RNA hybridization, SplintR is far more efficient in terms of
reaction kinetics. Thus, after a comparative trial (see Reaction System Optimization section), we chose SplintR as
the ligase used in the system.

![](https://static.igem.wiki/teams/4664/wiki/experiment/experiment/experiment/screen-shot-2023-10-09-at-10-12-40-pm.png)

#### RCA Process

The first step in RCA is the annealing of the miRNA to the padlock's miRNA binding sites. Then, SplintR ligase
circularises the originally linear-bent padlock. Phi29 DNA polymerase binds to the padlock-miRNA duplex and
initiates the amplification of a DNA chain complementary to the padlock (the amplicon).

![](https://static.igem.wiki/teams/4664/wiki/experiment/experiment/experiment/screen-shot-2023-10-09-at-10-14-05-pm.png)

### Cas12a

The enzyme Cas12a is a component of the CRISPR-Cas system, a potent tool for precise genetic modification. Unlike
Cas9, Cas12a locates its target DNA sequence using a single RNA guide molecule. Cas12a is used for recognizing the
domain, binding to target DNA, and carrying out cleavage.
In our detection method, Cas12a is coupled with CRISPR-RNA (crRNA) to form the Cas12a-crRNA ribonucleoprotein
complex (RNP). The role of the crRNA is to guide the cas12a enzyme to the target sequence (the RCA amplicon). The
RNP ‘scans’ the DNA in order to find a match with the crane sequence, after which it undergoes a conformational
change to activate its nuclease.

#### Trans-Cleavage

The trans-cleavage of Cas12a is nonspecific. Thus, it cleaves both the RCA amplicon, the padlock, and the
fluorophore-quencher complex which will be explained shortly. When there is an excess of padlocks or RCA amplicon,
trans-cleavage would preferentially cut these instead of reporters. When there is an excess of reporters, false
positives may also occur. Therefore it is important to regulate the reporter-padlock ratio.

#### Cis-Cleavage

Cis-cleavage of cas12a is specific, thus it only cleaves the RCA amplicon. Cis-cleavage amplicons are
complementary to the padlocks, and thus could serve as primers for initiating secondary RCA (i.e. the same process
as described in the RCA section, only with amplicons in place of miRNAs). Digestion of longer amplicon sequences
(long cuts) into shorter amplicons (short cuts) are primarily due to cis-cleavages.

### Intermediary Assays

Both the three-step and two-step assays are tandem combinations of RCA and Cas12a’s fluorogenic readout. However,
in the three-step method, ligation and rolling circle amplification (RCA) are separate, and the dye SYBR Green II
is added (commonly used to stain RNA/ssDNA for qPCR analysis). In the two-step method, RCA and ligation steps are
combined, and the fluorogenic readout is solely from the fluorophore-quencher reporting system.

#### Cis-Cleavage

Cis-cleavage of cas12a is specific, thus it only cleaves the RCA amplicon. Cis-cleavage amplicons are
complementary to the padlocks, and thus could serve as primers for initiating secondary RCA (i.e. the same process
as described in the RCA section, only with amplicons in place of miRNAs). Digestion of longer amplicon sequences
(long cuts) into shorter amplicons (short cuts) are primarily due to cis-cleavages.

### MB-ERC2

#### Schematics

The miRNA biomarker-based exponential RCA Cas12a/CRISPR system combines rolling circle amplification with Cas12a
cleavages. The overall reaction schematics are shown below.

![](https://static.igem.wiki/teams/4664/wiki/experiment/experiment/experiment/screen-shot-2023-10-09-at-10-24-33-pm.png)

#### Detection Mechanism

Our reporter is composed of a fluorophore linked to a quencher molecule. While the two are connected, the
quencher absorbs energy from the fluorophore, thus preventing it from emitting fluorescence. But when cas12a
trans-cleavage acts on the quencher-fluorophore and severs their link, the fluorophore would start giving out
fluorescence. Thus, the fluorescence level is used to quantify the concentration of miRNA biomarkers in the sample
tested.

#### Reaction System Optimization

* Ribonucleoprotein (RNP) Concentration: Cas12a cleavages significantly influence the outcome of the reactions.
  Cis-cleavages are important for the initiation of secondary RCAs, thus promoting exponential amplification.
  Trans-cleavages are vital in terms of expression of the fluorescence signals. Thus, it is important to determine
  the optimal amount of RNP required to best enhance reaction kinetics. Our standard RNP concentration is 1 nM,
  thus, we tested the MB-ERC2 system with varied RNP concentrations of NC, 0.5 nM, 2.5 nM, 4 nM, and 5 nM.
* Phi 29 Polymerase Concentration: Phi29 polymerase is important in terms of facilitating the amplification in
  RCA (i.e. synthesis of the DNA strand complementary to the padlock). On the basis of our standard concentration
  (0.1 U/µl), we tested NC, 0.05 U/µl, 0.15 U/µl, 0.2 U/µl, and 0.5 U/µl.
* Padlock Probe (PLP) Concentration: as previously mentioned in the Cas12a section, PLP-reporter ratio is vital
  to the experiment outcome. Padlocks are essential in the step of rolling circle amplification (RCA). Starting
  from the standard 100 nM, we set up trials with PLP concentrations of NC, 50 nM, 75 nM, 150 nM, and 200 nM.
* Reporter Concentration: PLP-reporter ratio is essential to the results of the assay reaction, moreover,
  reporters generate the fluorescence signals on which our analytical hardware is solely based. We investigated
  concentrations of 0.5 µM, 1 µM, 2 µM, and 5 µM (standard is 10 µM).
* Padlock Probe Sequence: in order to determine the PLP sequence that yields the best reaction kinetics, we
  designed: 1. padlocks with the crRNA recognition site at different locations, and; 2. padlocks with single,
  double, and triple mismatches at the miRNA binding sites. We have 18 padlocks for miR-223 in total.
* BSA Concentration: different amounts of BSA were added to the reaction system. We used 0.1 mg/mL, 0.3 mg/mL,
  and 0.4 mg/mL (standard 0.2 mg/mL).
* Synthetic short-cuts: according to He et al. (2019), RCA amplicons of different lengths have different effects
  on reaction kinetics when put into the reaction system to initiate secondary RCA. This is partly due to the fact
  that longer amplicons could form linear duplexes with un-ligated padlocks. We created a synthetic short-cut
  (short amplicon), and put the short-cuts in place of miRNA into the reaction system. Results show that
  short-cuts do in fact enhance secondary RCA, for short-cut results are markedly higher than ordinary MB-ERC2
  fluorescence.
* SplintR and T4 Ligase: According to Jin et al. (2016), SplintR ligase yields significantly better performance
  than T4 ligase. Thus, we designed this experiment, comparing 5 U/20 µl SplintR with 40 U/20 µl T4 ligase.
* SplintR ligase concentration: SplintR ligase is extremely important for initiating the RCA process. We
  investigated concentrations of 10 U/20 µl, 20 U/20 µl, 25 U/20 µl, and 50 U/20 µl (standard 5 U/20 µl).
* Buffer selection: we investigated the following buffer combinations: 1. Buffer 2.1 (NEB) + BSA; 2. SplintR
  buffer + BSA, and; 3. Phi29 buffer + BSA. The SplintR/BSA combination yielded the best results.

## Methods

---

### Polyadenylation and Reverse Transcription

We prepared a cDNA synthesis reaction for each RNA sample that can be analysed by PCR. Also, to determine the
absolute level of an miRNA using a standard curve, it’s required to generate cDNA from dilutions prepared from a
known concentration of synthetic miRNA.

In an RNase-free tube, combine the following reagents:

Table: Ploy(A) / cDNA Synthesis Reaction

| **Reagent** | **Volume (μl)** |
| --- | --- |
| mRQ Buffer (2x) | 5 |
| RNA sample (0.25-8μg ) | 3.75 |
| mRQ Enzyme | 1.25 |
| **Total Volume** | **10** |

In a thermal cycler, incubate the tube for 1 hour at body temperature, then terminate at 85ºC for 5
minutes.Moreover, add DEPC water to control the volume so that the cDNA would be ready for miRNA quantification
protocols.

Table: U6 qPCR Reaction

| **Reagent** | **Volume (μl)** |
| --- | --- |
| ddH2O | 9 |
| TB Green Advantage Premix (2X) | 12.5 |
| ROX Dye (50X) | 0.5 |
| U6 Forward primer (10μM) | 0.5 |
| U6 Reverse Primer (10μM) | 0.5 |
| cDNA | 2.0 |
| **Total Volume** | **25** |

Table: Sample qPCR Reaction

| **Reagent** | **Volume (μl)** |
| --- | --- |
| ddH2O | 9 |
| TB Green Advantage Premix (2X) | 12.5 |
| ROX Dye (50X) | 0.5 |
| miRNA-specific primer (10μM) | 0.5 |
| mRQ 3’Primer (10μM) | 0.5 |
| cDNA | 2.0 |
| **Total Volume** | **25** |

The portions are then transferred into 8-row tube distribution. The remaining cDNA is stored in a -20ºC fridge.

## Three Step Assay

---

Denaturation

| **Mix 1** | **2.2μl** |
| --- | --- |
| Padlock (100 nM) | 1 µl |
| miRNA | 1 µl |
| SplintR buffer | 0.2 µl |

80 ºC 5 min, 26 ºC 1 min

Ligation System (10 µl)

| **Mix 2** | **10μl** |
| --- | --- |
| Mix 1 | 2.2 µl |
| SplintR buffer | 0.8 µl |
| SplintR ligase | 0.25 µl |
| DEPC Water | 6.75 µl |

PCR machine, 37 ºC 2h & 65 ºC 10 min, 10 µl system

Reaction System (Mix)

| **Mix** | **20 µl total** |
| --- | --- |
| Phi 29 buffer | 2 µl |
| Phi 29 polymerase | 0.4 µl |
| BSA | 0.2 µl |
| dNTPs | 0.8 µl |
| 10x SYBR Green II | 2 µl |
| DEPC Water | 12.6 µl |
| Mix 2 | 2 µl |

37 ºC 2h, 65 ºC 10 min

Final Step

Add 5 µl RNP & 2 µl reporter:

| **Mix 2** | **20 µl** |
| --- | --- |
| RNP | 5 µl |
| reporter | 2 µl |

## RNP Preparation

| **Component** | **Volume** |
| --- | --- |
| Cas12a | 1 µl |
| crRNA | 1.5 µl |
| Buffer 2.1 | 0.5 µl |
| DEPC water | 2 µl |

## Two Step Assay

---

Denaturation

| **Components** | **Volume** |
| --- | --- |
| miRNA | 2 µl |
| padlock | 2 µl |
| SplintR buffer | 0.4 µl |

80 ºC 5 min, 26 ºC 1 min, 5 µl system

Ligation-RCA System

Then transfer to ligation-RCA system (a total of 20µl system) mix 2:

| **Mix 1** | **4.4μl** |
| --- | --- |
| Mix 1 | 4.4 µl |
| dNTP | 0.8 µl |
| BSA | 0.2 µl |
| Phi29 Polymerase | 0.2 µl |
| SplintR ligase | 0.5 µl |
| dd water | 12.3 µl |
| SplintR buffer | 1.6 µl |

37 ºC 2h & 65 ºC 10 min, 20 µl system

Final Step

Add 5 µl RNP & 2 µl reporter (10µM):

| **Mix 2** | **20 µl** |
| --- | --- |
| RNP | 5 µl |
| reporter | 2 µl |

## MB-ERC2(EXTRA-CRISPER)

Denaturation

| **Components** | **Per portion** |
| --- | --- |
| miRNA | 2 µl |
| padlock | 2 µl |
| SplintR buffer | 0.4 µl |

80 ºC 5 min, 26 ºC 1 min

Mix

| Components | Per portion |
| --- | --- |
| dNTP | 0.8 µl |
| RNP | 0.2 µl |
| SplintR ligase | 0.2 µl |
| BSA | 0.2 µl |
| Phi29 Polymerase | 0.2 µl |
| reporter | 0.2 µl |
| SplintR buffer | 1.6 µl |
| DEPC water | 12.2 µl |

## References

Jin, J., Vaud, S., Zhelkovsky, A., Pósfai, J., & McReynolds, L. A. (2016). Sensitive and specific miRNA detection
method using SplintR Ligase. Nucleic Acids Research, 44(13), e116. <https://doi.org/10.1093/nar/gkw399>

He, Y., Wen, Y., Tian, Z., Hart, N. T., Han, S., Hughes, S. J., & Zeng, Y. (2023b). A one-pot isothermal
Cas12-based assay for the sensitive detection of microRNAs. Nature Biomedical Engineering.
<https://doi.org/10.1038/s41551-023-01033-1>

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## Results Overview

In the journey of our iGEM project, our team has undertaken a series of experiments and simulations, pushing the
boundaries of synthetic biology to address the detection of COPD.

While we reached notable milestones, our journey was filled with challenges. From the initial absence of a signal
to pinpointing root causes, from system optimization to aligning our work with real-world applications, we
confronted various hurdles. Each challenge offered invaluable learning experiences, strengthening our resilience and
adaptability as researchers.

## RT-qPCR

First, we conducted RT-qPCR trials, which will later be used as comparison groups for results obtained from
MB-ERC2. Experiments were carried out for miR-1274a and miR-223 at concentrations of 100 pM, 10 pM, 1 pM, 500 fM,
100 fM, and 20 fM. Our goal was to determine the success of our selected miRNA biomarkers in an in vitro reaction.

RT-qPCR (Reverse Transcription Quantitative Real-time Polymerase Chain Reaction), is a combination of RT-PCR and
qPCR methods, and is commonly employed for the detection and quantification of RNA. The procedure involves the
enzyme reverse transcriptase converting total RNA or messenger RNA (mRNA) to complementary DNA (cDNA). This cDNA is
then amplified and used in quantitative PCR (qPCR) or real-time PCR to detect specific targets. This PCR method
utilises a number of fluorescent chemicals to quantify the amount of DNA at each cycle in real-time.

After the RT-qPCR trials, we excluded miR-1274a from future experiment plans due to its poor performance with
RT-qPCR. U6 represents control in the following results figures.

### U6 Trials: baseline/negative control for RT-qPCR

Figure 1 displays U6 RT-qPCR results at concentrations of 100 pM, 10 pM, 1 pM, 500 fM, 100 fM, and 20 fM.

![](https://static.igem.wiki/teams/4664/wiki/content/results/img01.png)

*Figure 1. RT-qPCR for U6.*

## miRNA RT-qPCR results

Figures 2 and 3 show the RT-qPCR results for miR-1274a and miR-223, respectively. A significantly lower
fluorescence level for miR-1274a is already observable, and will be further demonstrated in the by-concentration
charts.

![](https://static.igem.wiki/teams/4664/wiki/content/results/img02.png)

*Figure 2. RT-qPCR for miR-1274a.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img03.png)

*Figure 3. RT-qPCR for miR-223.*

### Concentration-based Comparison of U6, miR-1274a, and miR-223

Figures 4 to 9, as shown below, are comparisons of U6, miR-1274a, and miR-223 fluorescence levels for the various
concentrations used. miR-223 markedly outperforms both U6 and miR-1274a. It is evident that miR-1274a often yields
performances similar to or even worse than the negative control U6 group. Thus, we excluded miR-1274a from future
experiments.

![](https://static.igem.wiki/teams/4664/wiki/content/results/img04.png)

*Figure 4. 100 pM U6, miR-1274a, and miR-223.*

A paired T-test was performed on miR-223 and U6. The two-tailed P value is under 0.0001, which is extremely
statistically significant according to conventional criterion.

We also performed a paired T-test on miR-1274a and U6. P value equals 0.7835, meaning that by conventional
criterion the difference of the two groups is not statistically significant.

For information about these two T-tests, see Tables 1 and 2.

| Group | miR-223 | U6 |
| --- | --- | --- |
| Mean | 2233.9849 | 2184.1433 |
| SD | 260.7545 | 177.8525 |
| SEM | 28.9727 | 19.7614 |
| N | 81 | 81 |

*Table 1. miR-223 and U6 (100 pM)*

| Group | miR-1274a | U6 |
| --- | --- | --- |
| Mean | 2182.8416 | 2184.1433 |
| SD | 199.8611 | 177.8525 |
| SEM | 22.2068 | 19.7614 |
| N | 81 | 81 |

*Table 2. miR-1274a and U6 (100 pM)*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img05.png)

*Figure 5. 10 pM for U6, miR-1274a, and miR-223.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img06.png)

*Figure 6. 1 pM for U6, miR-1274a, and miR-223.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img07.png)

*Figure 7. 500 fM for U6, miR-1274a, and miR-223.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img08.png)

*Figure 8. 100 fM for U6, miR-1274a, and miR-223.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img09.png)

*Figure 9. 20 fM for U6, miR-1274a, and miR-223.*

## Intermediary Assays

Intermediary assays, or combinations of ligation, RCA, Cas12a, and qPCR, were carried out in order to investigate
the effectiveness of RCA-Cas12a, on which our MB-ERC2 assay is based. Both the three-step and two-step assays are
tandem combinations of RCA and Cas12a’s fluorogenic readout. However, in the three-step method (Figure 10a),
ligation and rolling circle amplification (RCA) are separate, and the dye SYBR Green II is added (commonly used to
stain RNA/ssDNA for qPCR analysis). In the two-step method (Figure 10b), RCA and ligation steps are combined, and
the fluorogenic readout is solely from the fluorophore-quencher reporting system. Significant differences between
the experimental groups and NC can already be observed.

![](https://static.igem.wiki/teams/4664/wiki/content/results/img10.png)
![](https://static.igem.wiki/teams/4664/wiki/content/results/img11.png)

*Figure 10. Two step and three step assays mechanism.*

## Two Step Assay Results

Figures 11 and 12 are results from the two-step assay (RCA-ligation + CRISPR), with miRNA concentrations of 100 pM,
10 pM, 1 pM, 500 fM, 100 fM, and 20 fM. The two step assay is similar to our final MB-ERC2 system, except that the
CRISPR step is still separate from the ligation-RCA step, and is non-isothermal. Markedly high results were obtained
for most concentrations tested.

A paired T-test was performed for NC and 20 fM two step. The P value is less than 0.0001, indicating statistically
insignificant difference.

Table 3 shows the data used in the T-test.

Group | 20 fM | NC | || Mean | 2121.4060 | 2014.9348 |
| SD | 34.3229 | 2.7662 |
| SEM | 6.8646 | 0.5532 |
| N | 25 | 25 |

*Table 3. 20 fM vs. NC (Two Step).*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img12.png)

*Figure 11. Two-Step assay 10 pM & 100 pM.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img13.png)

*Figure 12. Two-step assay 1 pM, 500 fM, 100 fM, and 20 fM result (x-axis is cycle, time per cycle is 10
minutes).*

## Three Step Assay

The three step assay is a tandem combination of ligation, RCA, and CRISPR. Experiments were performed at
concentrations of 500 fM, 100 fM, and 20 fM, all of which yields significantly higher fluorescence levels than the
NC.

A paired T-test was carried out for 20 fM vs. NC. P value is smaller than 0.0001, indicating statistically
significant difference. Data is shown in Table 4.

Group | 20 fM | NC | || Mean | 2153.744 | 2041.2648 |
| SD | 51.6162 | 5.3643 |
| SEM | 10.3232 | 1.0729 |
| N | 25 | 25 |

*Table 4. Three step 20 fM vs. NC.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img14.png)

*Figure 13. Three-Step assay 500 fM, 100 fM, and 20 fM.*

## MB-ERC2

Our miRNA biomarker-based exponential RCA Cas12a/CRISPR (MB-ERC2) system combines rolling circle amplification with
Cas12a cleavages. Figures 14-17 are results from MB-ERC2 experiments, a one-pot isothermal reaction system used to
detect miRNA biomarkers at low, in vivo-level concentrations. Significant differences could be observed between the
negative control and experiment groups, even for low concentrations such as 20 fM (close to miRNA concentrations in
vivo). Results from various optimization experiments are also included (Figures 18-36). Please see more on the
Experiments page.

## Basic MB-ERC2 Reactions

Figures 14-16 are the basic reactions for MB-ERC2 (based on EXTRA-CRISPR from He et al. (2023)). miRNA
concentrations include 1 nM, 100 pM, 10 pM, 1 pM, 500 fM, 100 fM, and 20 fM. Figure 17 is a comparison of no RNP vs.
regular MB-ERC2 reactions, intended to demonstrate the importance of Cas12a cleavage in the reaction system.

A paired T-test was performed on EC-20 fM and NC. The P value is less than 0.0001, indicating an extremely
statistically significant difference. Table 5 is data from the test.

Group | EC 20 fM | NC | || Mean | 2088.3783 | 2012.0552 |
| SD | 36.3071 | 4.5236 |
| SEM | 7.5705 | 0.9432 |
| N | 23 | 23 |

*Table 5. T-test for EC 20 fM & NC.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img15.png)

*Figure 14. EXTRA-CRISPR 100 pM, 1 nM (x-axis is cycle, time per cycle is 10 minutes).*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img16.png)

*Figure 15. EXTRA-CRISPR 500 fM, 1 pM, and 10 pM.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img17.png)

*Figure 16. 100 fM & 20 fM.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img18.png)

*Figure 17. NC RNP trials for 1 pM and 10 pM miRNA.*

## Optimization Reactions

Below are results from the various optimization experiments we performed in order to investigate the best
concentrations of reagents involved in the MB-ERC2 system, and to optimise the sequence of the padlock.

We performed the following optimization experiments:

1. Ribonucleoprotein (RNP) Concentration (Figures 21-23)
2. Phi 29 Polymerase Concentration (Figures 18-20)
3. Padlock Probe (PLP) Concentration (Figures 24-26)
4. Reporter Concentration (Figures 27-29)
5. Padlock Probe Sequence (Figure 35 & 36)
6. BSA Concentration (Figure 33)
7. Synthetic short-cuts (Figure 37)
8. SplintR and T4 Ligase (Figures 31 & 32)
9. SplintR ligase concentration (Figures 30 & 32)
10. Buffer selection (Figure 34)

Phi 29 Polymerase Concentration

Phi29 polymerase is important in terms of facilitating the amplification in RCA (i.e. synthesis of the DNA strand
complementary to the padlock). On the basis of our standard concentration (0.1 U/µl), we tested NC, 0.05 U/µl, 0.15
U/µl, 0.2 U/µl, and 0.5 U/µl

![](https://static.igem.wiki/teams/4664/wiki/content/results/img19.png)

*Figure 18. Phi29 Polymerase concentration optimization, 1 pM miRNA.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img20.png)

*Figure 19. Phi29 Polymerase concentration optimization, 10 pM miRNA.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img21.png)

*Figure 20. Final fluorescence levels for Phi29 concentration optimization.*

Ribonucleoprotein (RNP) Concentration

Cas12a cleavages significantly influence the outcome of the reactions. Cis-cleavages are important for the
initiation of secondary RCAs, thus promoting exponential amplification. Trans-cleavages are vital in terms of
expression of the fluorescence signals. Thus, it is important to determine the optimal amount of RNP required to
best enhance reaction kinetics. Our standard RNP concentration is 1 nM, thus, we tested the MB-ERC2 system with
varied RNP concentrations of NC, 0.5 nM, 2.5 nM, 4 nM, and 5 nM.

![](https://static.igem.wiki/teams/4664/wiki/content/results/img22-01.png)

*Figure 21. Ribonucleoprotein concentration optimization, 10 pM miRNA.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img23-01.png)

*Figure 22. RNP concentration optimization, 1 pM miRNA.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img24-01.png)

*Figure 23. Ribonucleoprotein concentration optimization final values.*

Padlock Probe (PLP) Concentration

The PLP-reporter ratio is vital to the experiment outcome (see Experiments page, Cas12a section). Padlocks are
essential in the step of rolling circle amplification (RCA). Starting from the standard 100 nM, we set up trials
with PLP concentrations of NC, 50 nM, 75 nM, 150 nM, and 200 nM.

![](https://static.igem.wiki/teams/4664/wiki/content/results/img25-01.png)

*Figure 24. Padlock Probe concentration optimization, 10 pM miRNA.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img26-01.png)

*Figure 25. Padlock Probe (PLP) concentration optimization, 1 pM miRNA.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img27-01.png)

*Figure 26. Padlock Probe concentration optimization final values.*

Reporter Concentration

Our reporter is composed of a fluorophore linked to a quencher molecule. While the two are connected, the quencher
absorbs energy from the fluorophore, thus preventing it from emitting fluorescence. But when cas12a trans-cleavage
acts on the quencher-fluorophore and severs their link, the fluorophore would start giving out fluorescence.
PLP-reporter ratio is essential to the results of the assay reaction, moreover, reporters generate the fluorescence
signals on which our analytical hardware is solely based. We investigated concentrations of 0.5 µM, 1 µM, 2 µM, and
5 µM (standard is 10 µM).

![](https://static.igem.wiki/teams/4664/wiki/content/results/img28.png)

*Figure 27. Reporter concentration optimization, 1 pM miRNA.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img29.png)

*Figure 28. Reporter concentration optimization, 10 pM miRNA.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img30.png)

*Figure 29. Final fluorescences levels for reporter concentration optimization.*

SplintR Ligase Concentration

SplintR ligase is extremely important for initiating the RCA process. We investigated concentrations of 10 U/20 µl,
20 U/20 µl, 25 U/20 µl, and 50 U/20 µl (standard 5 U/20 µl).

![](https://static.igem.wiki/teams/4664/wiki/content/results/img31.png)

*Figure 30. SplintR Ligase concentration optimization.*

T4 and SplintR Ligases

According to Jin et al. (2016), SplintR ligase yields significantly better performance than T4 ligase. Thus, we
designed this experiment, comparing 5 U/20 µl SplintR with 40 U/20 µl T4 ligase.

![](https://static.igem.wiki/teams/4664/wiki/content/results/img32.png)

*Figure 31. T4 vs. SplintR Ligase with 10 pM miRNA.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img33.png)

*Figure 32. SplintR & T4 final fluorescence levels.*

Bovine Serum Albumin Concentration

Different amounts of BSA were added to the reaction system. We used 0.1 mg/mL, 0.3 mg/mL, and 0.4 mg/mL (standard
0.2 mg/mL).

![](https://static.igem.wiki/teams/4664/wiki/content/results/img34.png)

*Figure 33. BSA concentration optimization.*

Buffer Optimization

We investigated the following buffer combinations: 1) Buffer 2.1 (NEB) + BSA; 2) SplintR buffer + BSA, and; 3)
Phi29 buffer + BSA. The SplintR/BSA combination yielded the best results.

![](https://static.igem.wiki/teams/4664/wiki/content/results/img35.png)

*Figure 34. Buffer selection.*

Padlock Sequence Optimization

In order to determine the PLP sequence that yields the best reaction kinetics, we designed: 1) padlocks with the
crRNA recognition site (responsible for binding with the crRNA in RNP and activating Cas12a cleavages) at different
locations, and; 2) padlocks with single, double, and triple mismatches at the miRNA binding sites. We have 18
padlocks for miR-223 in total.

![](https://static.igem.wiki/teams/4664/wiki/content/results/img36.png)

*Figure 35. Padlocks 1-4 with 10 pM miRNA and optimal reaction system.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img37.png)

*Figure 36. Padlocks M1-14 optimization.*

Synthetic Short-cuts

We created a synthetic short-cut (short amplicon), which anneals to the padlock and initiates secondary RCA
reactions. The short-cuts were then put in place of miRNA into the reaction system.

A paired T-test was also carried out for the 100 fM short-cut vs. NC. P value is less than 0.0001, meaning
statistically significant difference. Data is shown in Table 6.

Group | Short-cut 100 fM | NC | || Mean | 2332.2464 | 2050.9732 |
| SD | 102.5636 | 2.9386 |
| SEM | 20.5127 | 0.5877 |
| N | 25 | 25 |

*Table 6. T-test for 100 fM short-cut vs. NC.*

![](https://static.igem.wiki/teams/4664/wiki/content/results/img38.png)

*Figure 37. Synthetic short-cuts.*

## References

Jin, J., Vaud, S., Zhelkovsky, A., Pósfai, J., & McReynolds, L. A. (2016). Sensitive and specific miRNA detection method using SplintR Ligase. Nucleic Acids Research, 44(13), e116. <https://doi.org/10.1093/nar/gkw399>

He, Y., Wen, Y., Tian, Z., Hart, N. T., Han, S., Hughes, S. J., & Zeng, Y. (2023b). A one-pot isothermal Cas12-based assay for the sensitive detection of microRNAs. Nature Biomedical Engineering. <https://doi.org/10.1038/s41551-023-01033-1>

Koch, M., Butt, T., Wudong, G., Li, X., Chen, Y., Tan, D. W., & Liu, G. G. (2019).
Characteristics and health burden of the undiagnosed population at risk of chronic
obstructive pulmonary disease in China. BMC Public Health, 19(1).
<https://doi.org/10.1186/s12889-019-8071-8>

Zieliński, J., & Bednarek, M. (2001). Early detection of COPD in a High-Risk population using spirometric screening. Chest, 119(3), 731–736. <https://doi.org/10.1378/chest.119.3.731>

Feldman, W. B., Bloomfield, D., Beall, R. F., & Kesselheim, A. S. (2022). Patents And Regulatory Exclusivities On Inhalers For Asthma And COPD, 1986–2020. Health Affairs, 41(6), 787–796. <https://doi.org/10.1377/hlthaff.2021.01874>

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