Part:BBa_K5087010
SynF
Introduction
SynF is a forward primer specifically designed to amplify a fragment of synthetic DNA in an RPA (Recombinase Polymerase Amplification). It is used to create a positive control assay within the SynLOCK system.
This primer contains a T7 promoter to enable in vitro transcription of the amplified product into RNA, preparing it for SHERLOCK detection with Cas13 proteins.
Biology & Usage
Controls are essential for conducting robust scientific research. In SHERLOCK tests, the synthetic DNA template acts as a positive control, ensuring that the materials used are of the appropriate quality and that the entire process is functioning as expected. Additionally, using synthetic DNA allows researchers to practice and refine the SHERLOCK detection method without wasting valuable nucleic acid samples. [1]
To prepare the DNA template, an RPA reaction must be performed to introduce the sequence recognized by T7 RNA polymerase, enabling the synthesis of RNA that can be detected by Cas13 proteins in the SHERLOCK assay.
RPA Reaction
Recombinase Polymerase Amplification (RPA) is an isothermal nucleic acid amplification technique that operates at a temperature range of 37–42°C, distinguishing it from traditional PCR methods that require thermal cycling for denaturation and annealing of DNA [2].
RPA relies on three essential types of proteins: a recombinase, single-stranded DNA binding proteins (SSBs), and a strand-displacing DNA polymerase.
The process begins when the recombinase protein binds to a primer (about 30–35 nucleotides long) that matches the target DNA sequence. This complex then searches for homologous sequences in double-stranded DNA and initiates strand invasion. The SSBs stabilize the displaced strand to prevent primer dissociation, while the strand-displacing DNA polymerase extends the primer, resulting in exponential amplification of the target sequence [3].
Figure 1. The RPA (Recombinase Polymerase Amplification) Mechanism
SHERLOCK Method
The SHERLOCK platform is a modern synthetic biology tool that utilizes the properties of the Cas13a protein, an enzyme from the Nobel Prize-winning CRISPR-Cas system. The Cas13a protein is guided with high specificity to the target sequence using crRNA.
The crRNA molecule is crucial for the assay's specificity. It consists of a direct repeat (DR) sequence and a spacer sequence that is complementary to the target. The crRNA molecule is designed to uniquely identify the organism by targeting the Internal Transcribed Spacer (ITS) sequence in its genome.
First, the Cas13a protein binds to the organism's genetic material, which was previously amplified using RPA and transcribed into RNA. Once bound, the Cas13a protein is activated and exhibits a “collateral” RNase activity, meaning it non-specifically cleaves nearby single-stranded RNA molecules [1].
This activity can be used in assays by including synthetic RNA probes tagged with a fluorescent reporter and a quencher in the reaction mixture. A fluorescent signal indicates that the reporters have been cleaved by Cas13, confirming the presence of the DNA target in the sample. The SHERLOCK method can also be used with Lateral Flow Assays (LFA).
Figure 2. The SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) Method Mechanism
Part Performance
As mentioned earlier, the synthetic DNA positive control is crucial for all SHERLOCK tests. It ensures reliability and accuracy in the detection process, making it an essential component of any SHERLOCK assay.
Experimental validation
In this test, we aimed to evaluate the design of our PrymcrRNA1 and PrymcrRNA2. The synDNA1 + syncrRNA sample served as a positive control to confirm that the materials were of appropriate quality and that the procedure was executed correctly.
Both the RPA and SHERLOCK reactions were prepared following the guidelines provided by Kellner et al. [1]. In our tests we used the LwaCas13a protein.
Figure 3. Fluorescence readout results demonstrate the performance of various part combinations from our toolkit. The bright green line (indicated as "synDNA1 syncrRNA") shows the SHERLOCK reaction where the synDNA1 template was first amplified via RPA using SynF and SynR primers and then detected using SynCrRNA.
Conclusions: Both PrymcrRNA designs were successful in detecting algal DNA. The synDNA + syncrRNA positive control functioned as expected, producing a strong fluorescence signal. When a mismatch between the DNA template and crRNA was introduced (synDNA1 + PrymCrRNA1/2), no signal was detected, confirming that the positive control is working properly.
Parts Compatible with SynF
For the positive control to function properly, SynF must be used together with SynR (BBa_K5087011), the synthetic DNA template [1], and SynCrRNA (BBa_K5087024).
PrymFlow
Introduction to the Lateral Flow Assay
The SHERLOCK method, like other CRISPR/Cas-based detection techniques, is compatible with a Lateral Flow readout format. The Lateral Flow Test is known for its speed, simplicity, and ease of interpretation. We aimed to adapt the previously optimized SHERLOCK components for use with Lateral Flow dipsticks, resulting in the development of the PrymFlow test for detecting the presence of Prymnesium parvum.
LFA Result Interpretation
If the target is present, the T-line becomes visible, indicating a positive result. Meanwhile, the C-line serves as a control that is less visible when greater amounts of the target sequence are present. Gold nanoparticles (GNPs) with anti-FAM antibodies provide the visual indication of the test result.
WHAT HAPPENS WHEN Prymnesium parvum IS NOT PRESENT IN THE WATER SAMPLE?
Streptavidin, immobilized on the C-line, captures the biotin-labeled ends of the intact reporters. The reporters are captured on the C-line, and the binding of gold nanoparticles (GNPs) conjugated with anti-FAM antibodies makes only the C (control) line visible.
AND WHEN IT IS PRESENT?
The reporters are cleaved by the activated Cas protein. Consequently, gold nanoparticles (GNPs) with anti-FAM antibodies capture the FAM-labeled fragments, which then bind to the anti-anti-FAM antibody immobilized on the test line (T-line), producing a strong signal. The presence of the T-line indicates the presence of Prymnesium parvum. Some intact reporters might remain in the mix (the amount depends on how many target DNA molecules were present in the sample, influencing the ratio of activated to non-activated Cas13 protein and the number of cleaved reporter molecules). As a result, a weak control line (C-line) is visible.
Figure 4. Explanation of the lines visible on the Lateral Flow test during a negative and positive result.
The LFA tests were conducted following the protocol, which can be accessed here.
Figure 5. 8. SynF-SynR + SynCrRNA (Positive control).
Test sample 8, containing the SynF primer, produced a 100% positive result, indicated by the presence of only the T-line (upper band) on the strip.
Note: The remaining LFA strips pertain to other BioBrick parts or are not relevant to the conclusions presented here.
Sequence
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 30
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 24
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 30
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 30
- 1000COMPATIBLE WITH RFC[1000]
Biosafety
We used the Asimov's tool — Kernel — to check the sequence's safety with the Biosecurity Sequence Scanner. The results showed no flagged sequences, confirming that this part is safe to use.
Sequence source and Design
This sequence was introduced by Kellner et al. [1]. The SynF corresponds to the sequence named “Synthetic DNA 1 RPA forward primer”.
Resources
- [1] Kellner, Max J., Jeremy G. Koob, Jonathan S. Gootenberg, Omar O. Abudayyeh, and Feng Zhang. “SHERLOCK: Nucleic Acid Detection with CRISPR Nucleases.” Nature Protocols 14, no. 10 (October 2019): 2986–3012. https://doi.org/10.1038/s41596-019-0210-2
- [2] Lobato, I. M., & O'Sullivan, C. K. (2018). Recombinase polymerase amplification: Basics, applications and recent advances. Trends in Analytical Chemistry: TRAC, 98, 19–35. https://doi.org/10.1016/j.trac.2017.10.015
- [3] Ruichen Lv, Nianhong Lu, and Junhu Wang et al. Recombinase Polymerase Amplification for Rapid Detection of Zoonotic Pathogens: An Overview. Zoonoses, 2(1). DOI: 10.15212/ZOONOSES-2022-0002
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