Figure 1. Overall concept of our project. “RCA” is the abbreviation of “Rolling Circle Amplification”. “AuNPs” is the abbreviation of “Gold Nanoparticles”. “RT-RPA” is the abbreviation of “Reverse Transcription-Recombinase Polymerase Amplification”. “PCRD” is a kind of lateral flow strip.

The ultimate objective of our project is attempting to employ human serum as the sample for both gold nanoparticle (AuNPs)-based colorimetric assay [1] and lateral flow strip testing [2]. However, at the current stage, we are unable to obtain serum samples from individuals due to permissions from relevant government institutions (see page implementation for our planned use of human specimens). Consequently, to validate the two pathways outlined in Figure 1, we synthesized circular RNA with an identical sequence to that found in human serum. Initially, the circularized product was amplified using either rolling circle amplification (RCA) or reverse transcription-recombinase polymerase amplification (RT-RPA). Subsequently, the amplified products were tested using gold nanoparticle-based colorimetric assay or lateral flow test, respectively.

Figure 2. The seven experimental pathway, labeled as A-G.

***Click any interested part of design or line to see different experimental pathway***

We designed seven different experimental pathways (Figure 2) to validate whether the colorimetric assay and lateral flow strip would function as expected. All the A-G pathways are categorized into three sections: “CircRNA synthesis”, “Gold nanoparticle-based colorimetric assay” and “Lateral flow test.” The following description outlines the objective and design of each pathway.

    CircRNA synthesis

Path A: CircRNA Synthesis

Goal: Synthesis of our target circRNA, hsa_circ_0004771 and hsa_circ_0101802, from its complementary DNA (cDNA) sequence, Insert_0004771 (BBa_K4636041) and Insert_0101802 (BBa_K4636040), which we ordered from synthetic customized DNA oligonucleotide manufacturer service vendor (Integrated DNA Technologies).

Path: Sequence design → Cloning → PCR → IVT → Circularization → CircRNA enrichment

Design:

Figure 3. The experimental design, from IVT (In vitro transcription) to circRNA enrichment, of circRNA synthesis.

Sequence design: All the required DNA for each step should be designed before conducting the experiment. Below is a brief explanation of different sequences required in path A; please refer to the Parts page for more details.

Cloning: Inserts (Insert_0004771 (BBa_K4636041) and Insert_0101802 (BBa_K4636040)) are designed to ligate with vector.

PCR: Forward (BBa_K4636003 and BBa_K4636005) and reverse (BBa_K4636004 and BBa_K4636006) primers are designed to specifically amplify the inserts we ligated with vectors during cloning.

IVT: The modified T7 promotor binding sites are incorporated into Insert_0004771 (BBa_K4636041) and Insert_0101802 (BBa_K4636040) to facilitate T7 polymerase functionality.

Circularization: DNA oligo, known as splint (BBa_K4636013-BBa_K4636018), are designed to elevate the efficiency of RNA back splicing by forming a double-stranded RNA (dsRNA).

Cloning: We ligate cDNA sequences (insert) with the pUC19 vector, followed by the transfer of the vector into E. coli DH5-alpha. This cloning process ensures a continuous and abundant supply of the inserted sequences.

PCR: The abbreviation of “Polymerase Chain Reaction”. We extract and amplify cDNA sequences from plasmid utilizing colony PCR for use in the subsequent experiment.

IVT: The abbreviation of “In Vitro Transcription”. The cDNA sequences are transcribed into linear RNA sequences with the assistance of T7 polymerase. Followed by treating with DNase to degrade remaining cDNA template.

Circularization: Circularization of linear RNA includes three main steps as below.

1. Monophosphorylation: Monophosphorylation is crucial process prior to the back splicing step. It can be achieved by treating the RNA with pyrophosphatase (RppH), which removes the pyrophosphate group from the 5'-end of triphosphorylated RNA, ultimately yielding a 5'-monophosphate RNA. Followed by back splicing, the monophosphorylated RNA can form a covalent bond between the 5'- and 3'-ends [3].

2. Formation of dsRNA: Prior to the back-splicing step, we introduce a type of ssDNA oligo, known as splint. The splint has a complementary sequence to the linear form RNA. With this characteristic, it enhances the circularization ability of our RNA, by forming a double-stranded RNA [4].

3. Back-splicing: The monophosphrylated linear RNA with splint attached can finally form the circRNA we desired in the assistance of T4 RNA ligase 2. After the ligation process, the splint will be degraded by treating with DNase I [5].

Figure 4. A diagram shows how splint and T4 RNA ligase 2 help the formation of circRNA.

CircRNA enrichment: Given the circRNA's resistance to RNA exonuclease activity, we are able to remove any remaining linear RNA by treating the back-splicing products with RNase R [6]. Subsequently, we extract circRNA using an RNA clean-up system. This is followed by isothermal amplification (either RCA or RT-RPA) for colorectal cancer detection (via either Gold nanoparticle-based colorimetric assay or Lateral flow test).

Click to go Results A!

    Gold nanoparticle-based colorimetric assay

Figure 5. A diagram shows the mechanism of gold nanoparticle-based colorimetric assay.

If the RCA products (long-repeated single-stranded DNA) are present in the probe-conjugated gold nanoparticle solution, the solution will appear a red color; conversely, if the RCA products are absent in the probe-conjugated gold nanoparticles solution, the color of solution will turn purple [1]. (see page description for a detailed explanation of the principle)

Path B: Probe-Conjugated Gold Nanoparticle Synthesis

Goal: Preparation of probe-conjugated gold nanoparticles (AuNPs) for the colorimetric assay.

Path: Sequence design → AuNPs synthesis → Probe-conjugated AuNPs → Probe-conjugated AuNPs detection

Design:

Sequence design: All the required DNA for each step should be designed before conducting the experiment. Below is a brief explanation of sequences required in path B; please refer to the Parts page for more details.

Probe conjugated AuNPs: A kind of DNA oligo, known as probe (BBa_K4636007-BBa_K4636012), were designed to act as the bridge between gold nanoparticles and RCA product (long-repeated single-stranded DNA sequence).

AuNPs synthesis: According to our principle investigator, we could synthesize gold nanoparticles with diameter of approximately 13 nm for the colorimetric assay. Theoretically, 13 nm gold nanoparticles exhibit a peak in their spectrum at around 520 nm [7]. Therefore, our aim is to synthesize gold nanoparticles with a peak at approximately 520 nm and employ scanning electron microscopy (SEM) to double-check if the product aligns with our expectations.

Probe-conjugated AuNPs: Thiol-modified DNA probes [8] with sequence complementary to the RCA product are conjugated onto the surface of 13 nm gold nanoparticles.

Probe-conjugated AuNPs detection: The probe-conjugated gold nanoparticles are then utilized for detecting the long-repeated single-stranded DNA sequence (produced either by AELA-PCR or RCA).

Click to go Results B!

Path C: Assess the Functionality of Colorimetric Assay with AELA-PCR

Goal: Considering that the circularization of RNA might be a challenging task and due to time limitation, we aim to synthesize a long-repeated single-stranded DNA (ssDNA) sequence to mimic the RCA product.

Path: Sequence design → Cloning → PCR → Long-repeated dsDNA synthesis → AELA-PCR → Probe conjugated AuNPs detection

Design:

Figure 6. The experimental design of Path C.

Sequence design: All the required DNA for each step should be designed before conducting the experiment. Below is a brief explanation of different sequences required in path C; please refer to the Parts page for more details.

Cloning: Inserts (Insert_0004771 (BBa_K4636041) and Insert_0101802 (BBa_K4636040)) are designed to ligate with vector.

PCR: Forward (BBa_K4636003 and BBa_K4636005) and reverse (BBa_K4636004 and BBa_K4636006) primers are designed to specifically amplify the inserts we ligate with vectors during cloning.

Preparation of template for AELA-PCR: Forward (BBa_K4636036) and reverse (BBa_K4636037) primers are designed to specifically amplify the long-repeated inserts we ligate with vectors during preparation of template for AELA-PCR.

AELA-PCR: Forward (BBa_K4636038) and reverse (BBa_K4636039) primers are designed to specifically and asymmetrically amplify the long-repeated insert we produce during long-repeated dsDNA synthesis.

Cloning: We ligate cDNA sequences with the pUC19 vector, followed by the transfer of the vector into E. coli DH5-alpha. This cloning process ensures a continuous and abundant supply of the inserted sequences.

PCR: The abbreviation of “Polymerase Chain Reaction”. We extract and amplify cDNA sequences from plasmid utilizing colony PCR for use in the subsequent experiment.

Repeated insert sequence synthesis: We introduce two restriction enzymes to digest the PCR products. The resulting 3'- and 5' sticky ends on the inserts are then ligated, forming long-repeated insert sequences with the assistance of T4 DNA ligase.

Preparation of template for AELA-PCR: There are two main purposes we attempt to achieve. Firstly, incorporating additional sequences at the 3'- and 5'- ends of the long-repeated insert sequences, respectively, where the primers (BBa_K4636038 and BBa_K4636039) designed for AELA-PCR can bind. Secondly, amplifying the long-repeated insert sequences to act as the template for AELA-PCR. We introduce cloning again to achieve these two purposes.

AELA-PCR: The abbreviation of “Asymmetric Exponential and Linear Amplification-Polymerase Chain Reaction”. AELA-PCR is a form of asymmetric PCR, a technique that transform dsDNA into ssDNA. This is achieved by adding forward and reverse primer in a ratio of 20:1 and processing a specialized thermal profile during the process [9].

Probe-conjugated AuNPs detection: The long-repeated ssDNA sequences can be use as sample, mixed with probe-conjugated gold nanoparticle solution produced in Path B to test if the colorimetric assay functions as expected (Figure 5).

Click to go Results C!

Path D: Amplify CircRNA by RCA for Colorimetric Assay Detection

Goal: Amplify the synthesized circRNA by rolling circle amplification (RCA) and utilize the resulting product to evaluate the functionality of gold nanoparticle-based colorimetric assay.

Path: Sequence design → Cloning → PCR → IVT → Circularization → CircRNA enrichment → RCA → Probe-conjugated AuNPs detection

Design:

Sequence design: All the required DNA for each step should be designed before conducting the experiment. Below is a brief explanation of different sequences required in path D; please refer to the Parts page for more details

Cloning: Inserts (Insert_0004771 (BBa_K4636041) and Insert_0101802 (BBa_K4636040)) that we planned ligating with vector.

PCR: Forward (BBa_K4636003 and BBa_K4636005) and reverse (BBa_K4636004 and BBa_K4636006) primers are designed to specifically amplify the inserts we ligate with vectors during cloning.

IVT: The modified T7 promotor binding sites are incorporated into Insert_0004771 (BBa_K4636041) and Insert_0101802 (BBa_K4636040) to facilitate T7 polymerase functionality.

Circularization: DNA oligo, known as splint (BBa_K4636013-BBa_K4636018), are designed to elevate the efficiency of RNA back splicing by forming a double-stranded RNA (dsRNA).

RCA: Single-stranded DNA primers (BBa_K4636028-BBa_K4636035) are designed to facilitate the initiation of the isothermal amplification process.

Cloning: We ligate cDNA sequences with the pUC19 vector, followed by the transfer of the vector into E. coli DH5-alpha. This cloning process ensures a continuous and abundant supply of the inserted sequences.

PCR: The abbreviation of “Polymerase Chain Reaction”. We extract and amplify cDNA sequences from plasmid utilizing colony PCR for use in the subsequent experiment.

Figure 7. A diagram shows the experimental design, from IVT (In vitro transcription) to circRNA enrichment, of circRNA synthesis.

IVT: The abbreviation of “In Vitro Transcription”. The cDNA sequences are transcribed into linear RNA sequences with the assistance of T7 polymerase. Followed by treating with DNase to degrade remaining cDNA template.

Circularization: Circularization of linear RNA includes three main steps as below.

Monophosphorylation: Monophosphorylation is crucial process prior to the back splicing step. It can be achieved by treating the RNA with pyrophosphatase (RppH), which removes the pyrophosphate group from the 5'-end of triphosphorylated RNA, ultimately yielding a 5'-monophosphate RNA. Followed by back splicing, the monophosphorylated RNA can form a covalent bond between the 5'- and 3'-ends [3].

Formation of dsRNA: Prior to the back splicing step, we introduce a type of DNA oligo, known as splint. The splint has a complementary sequence to the linear form RNA. With this characteristic, it enhances the circularization ability of our RNA, by forming a double-stranded RNA [4].

Back-splicing: The monophosphrylated linear RNA with splint attached can finally form the circRNA we desired in the assistance of T4 RNA ligase 2. After the ligation process is done, the splint will be degraded by treating with DNase I [5].

Figure 8. A diagram shows how splint and T4 RNA ligase 2 help the formation of circRNA.

CircRNA enrichment: Given the circRNA's resistance to RNA exonuclease activity, we are able to remove any remaining linear RNA by treating the back splicing products with RNase R [6]. Subsequently, we extract circRNA using an RNA clean-up system.

RCA: The abbreviation of “Rolling Circle Amplification”. RCA is an isothermal amplification method that the reaction temperature of approximately 60-65°C. At first, the circRNA serves as template short DNA primers will annealed onto the complementary site. Subsequently, reverse transcriptase can amplify through continuous and repetitive reverse transcription along the circRNA, resulting in the formation of a long-repeated single-stranded complementary DNA [10].

Figure 9. A diagram shows how RCA amplify circular RNA by producing long repeated single-stranded complementary DNA.

Probe-conjugated AuNPs detection: The long-repeated single-stranded complementary DNA can be use as sample, mixed with probe-conjugated gold nanoparticle solution produced in Path B to test if the colorimetric assay functions as expected (Figure 5).

Click to go Results D!

    Lateral flow test

Figure 10. A diagram shows the mechanism of lateral flow strip (PCRD).

If our target biomarkers, hsa_circ_0004771 and hsa_circ_0101802, present in the RT-RPA reaction, the resulting products (double-stranded DNA with modified primers) can bind to the anti-DIG and anti-FAM capture antibodies on the test lines. Consequently, three lines (two test lines and one control line) will be observed on the lateral flow strip (PCRD). Conversely, if there are no biomarkers amplified by RT-RPA, only the control line will be visible on the PCRD [2]. (see page description for a detailed explanation of the principle)

Path E: Assess the Functionality of Lateral Flow Test by RPA Products

Goal: Considering that the circularization of RNA might be a challenging work and also due to the time limitation. We have come up with an experimental pathway that skips the part of in vitro transcription (IVT) and circRNA synthesis and directly tests the functionality of the lateral flow strip (PCRD) right after polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA).

Path: Sequence design → Cloning → PCR → RPA → PCRD

Design:

Sequence design: All the required DNA for each step should be designed before conducting the experiment. Below is a brief explanation of different sequences required in path E; please refer to the Parts page for more details.

Cloning: Inserts (Insert_0004771 (BBa_K4636041) and Insert_0101802 (BBa_K4636040)) that we planned ligating with vector.

PCR: Forward (BBa_K4636003 and BBa_K4636005) and reverse (BBa_K4636004 and BBa_K4636006) primers are designed to specifically amplify the inserts we ligate with vectors during cloning.

RPA: Forward (BBa_K4636019, BBa_K4636021, BBa_K4636023, BBa_K4636025 and BBa_K4636027) and reverse (BBa_K4636020, BBa_K4636022, BBa_K4636024 and BBa_K4636026) primers are designed to facilitate the initiation of isothermal amplification process.

PCRD: Some modifications should be incorporated to the RPA forward (BBa_K4636019, BBa_K4636021, BBa_K4636023, BBa_K4636025 and BBa_K4636027) and reverse (BBa_K4636020, BBa_K4636022, BBa_K4636024 and BBa_K4636026) primers for the function of lateral flow strip.

Cloning: We ligate cDNA sequences with the pUC19 vector, followed by the transfer of the vector into E. coli DH5-alpha. This cloning process ensures a continuous and abundant supply of the inserted sequences.

PCR: The abbreviation of “Polymerase Chain Reaction.” We extract and amplify cDNA sequences from plasmid utilizing colony PCR for use in the subsequent experiment.

RPA: The abbreviation of “Recombinase Polymerase Amplification”. RPA is an isothermal amplification method that the reaction temperature of approximately 37-42°C. In the beginning, modified primers recognize its complementary sequence and insert into the double-stranded DNA template by the strand-displacement activity of the recombinase (Figure 11.①) Subsequently, the single-stranded binding protein (SSB) will stabilize the displaced DNA strain (Figure 11.②). The recombinase then disassemble (Figure 11.③) to allow polymerase binding and elongation of the modified primer (Figure 11.④ and Figure 11.⑤) [11].

Figure 11. The mechanism of recombinase polymerase amplification (RPA).

PCRD: The RPA products serve as sample to test the functionality of PCRD.

Click to go Results E!

Path F: Conducting RT-RPA Using Linear RNA as Template for Lateral Flow Test

Goal: Resulting from our inability to find relevant literature on reverse transcription-recombinase polymerase amplification (RT-RPA) using circRNA as a template and also due to the time limitation. We come up with an experimental pathway to omit the circRNA synthesis step. Instead, we will proceed RT-RPA using linear RNA as a template to access the functionality of PCRD.

Path: Sequence design → Cloning → PCR → IVT → RT-RPA → PCRD

Design:

Sequence design: All the required DNA for each step should be designed before conducting the experiment. Below is a brief explanation of different sequences required in path F; please refer to the Parts page for more details.

Cloning: Inserts (Insert_0004771 (BBa_K4636041) and Insert_0101802 (BBa_K4636040)) that we planned ligating with vector.

PCR: Forward (BBa_K4636003 and BBa_K4636005) and reverse (BBa_K4636004 and BBa_K4636006) primers are designed to specifically amplify the inserts we ligate with vectors during cloning.

IVT: The modified T7 promotor binding sites are incorporated into Insert_0004771 (BBa_K4636041) and Insert_0101802 (BBa_K4636040) to facilitate T7 polymerase functionality.

RT-RPA: Forward (BBa_K4636019, BBa_K4636021, BBa_K4636023, BBa_K4636025 and BBa_K4636027) and reverse (BBa_K4636020, BBa_K4636022, BBa_K4636024 and BBa_K4636026) primers are designed to facilitate the initiation of isothermal amplification process.

PCRD: Some modifications should be incorporated to the RPA forward (BBa_K4636019, BBa_K4636021, BBa_K4636023, BBa_K4636025 and BBa_K4636027) and reverse (BBa_K4636020, BBa_K4636022, BBa_K4636024 and BBa_K4636026) primers for the function of lateral flow strip.

Cloning: We ligate cDNA sequences with the pUC19 vector, followed by the transfer of the vector into E. coli DH5-alpha.This cloning process ensures a continuous and abundant supply of the inserted sequences.

PCR: The abbreviation of “Polymerase Chain Reaction”. We extract and amplify cDNA sequences from plasmid utilizing colony PCR for use in the subsequent experiment.

IVT: The abbreviation of “In Vitro Transcription”. The cDNA sequences are transcribed into linear RNA sequences with the assistance of T7 polymerase. Followed by treating with DNase to degrade any remaining cDNA.

RT-RPA: The abbreviation of “Reverse Transcription-Recombinase Polymerase Amplification”. RT-RPA is an isothermal amplification method with a reaction temperature of approximately 37-42°C. The process can be divided into two parts: reverse transcription (RT) and recombinase polymerase amplification (RPA). Initially, the linear RNA synthesized during IVT is reverse transcribed into complementary DNA which serve as template in the following RPA process (Figure 12). Subsequently, modified primers recognize its complementary sequence and insert into the double-stranded DNA template through the strand-displacement activity of the recombinase (Figure 12.①) . The single-stranded binding protein (SSB) then stabilizes the displaced DNA strand (Figure 12.②). Finally, the recombinase dissociates (Figure 12.③) to allow polymerase binding and elongation of the modified primer (Figure 12.④ and Figure 12.⑤) [12] .

Figure 12. The mechanism of recombinase polymerase amplification (RPA).

PCRD: The RT-RPA products (double-stranded DNA with modified primers) serve as sample to test the functionality of PCRD.

Click to go Results F!

Path G: Amplify CircRNA Utilizing RT-RPA for Lateral Flow Test

Goal: Successfully processing reverse transcription-recombinase polymerase amplification (RT-RPA) using circRNA as template. Subsequently, use the resulting product to access the functionality of lateral flow strip (PCRD).

Path: Sequence design → Cloning → PCR → IVT → Circularization → CircRNA enrichment → RT-RPA → PCRD

Design:

Sequence design: All the required DNA for each step should be designed before conducting the experiment. Below is a brief explanation of different sequences required in path G; please refer to the Parts page for more details.

Cloning: Inserts (Insert_0004771 (BBa_K4636041) and Insert_0101802 (BBa_K4636040)) that we planned ligating with vector.

PCR: Forward (BBa_K4636003 and BBa_K4636005) and reverse (BBa_K4636004 and BBa_K4636006) primers are designed to specifically amplify the inserts we ligate with vectors during cloning.

IVT: The modified T7 promotor binding sites are incorporated into Insert_0004771 (BBa_K4636041) and Insert_0101802 (BBa_K4636040) to facilitate T7 polymerase functionality.

Circularization: DNA oligo, known as splint (BBa_K4636013-BBa_K4636018), are designed to elevate the efficiency of RNA back splicing by forming a double-stranded RNA (dsRNA).

RT-RPA: Forward (BBa_K4636019, BBa_K4636021, BBa_K4636023, BBa_K4636025 and BBa_K4636027) and reverse (BBa_K4636020, BBa_K4636022, BBa_K4636024 and BBa_K4636026) primers are designed to facilitate the initiation of isothermal amplification process.

PCRD: Some modifications should be incorporated to the RPA forward (BBa_K4636019, BBa_K4636021, BBa_K4636023, BBa_K4636025 and BBa_K4636027) and reverse (BBa_K4636020, BBa_K4636022, BBa_K4636024 and BBa_K4636026) primers for the function of lateral flow strip.

Cloning: We ligate cDNA sequences with the pUC19 vector, followed by the transfer of the vector into E. coli DH5-alpha. This cloning process ensures a continuous and abundant supply of the inserted sequences.

PCR: The abbreviation of “Polymerase Chain Reaction”. We extract and amplify cDNA sequences from plasmid utilizing colony PCR for use in the subsequent experiment.

Figure 13. A diagram shows the experimental design, from IVT (In vitro transcription) to circRNA enrichment, of circRNA synthesis.

IVT: The abbreviation of “In Vitro Transcription”. The cDNA sequences are transcribed into linear RNA sequences with the assistance of T7 polymerase. Followed by treating with DNase to degrade any remaining cDNA.

Circularization: Circularization of linear RNA includes three main steps as below.

Monophosphorylation: Monophosphorylation is crucial process prior to the back splicing step. It can be achieved by treating the RNA with pyrophosphatase (RppH), which removes the pyrophosphate group from the 5'-end of triphosphorylated RNA, ultimately yielding a 5'-monophosphate RNA. Followed by back splicing, the monophosphorylated RNA can form a covalent bond between the 5'- and 3'-ends [3].

Formation of dsRNA: Prior to the back splicing step, we introduce a type of DNA oligo, known as splint. The splint has a complementary sequence to the linear form RNA. With this characteristic, it enhances the circularization ability of our RNA, by forming a double-stranded RNA [4].

Back-splicing: The monophosphrylated linear RNA with splint attached can finally form the circRNA we desired in the assistance of T4 RNA ligase 2. After the ligation process is done, the splint will be degraded by treating with DNase I [5].

Figure 14. A diagram shows how splint and T4 RNA ligase 2 help the formation of circular RNA.

CircRNA enrichment: Given the circRNA's resistance to RNA exonuclease activity, we are able to remove any remaining linear RNA by treating the back splicing products with RNase R [6]. Subsequently, we extract circRNA using an RNA clean-up system.

RT-RPA: The abbreviation of “Reverse Transcription-Recombinase Polymerase Amplification”. RT-RPA is an isothermal amplification method with a reaction temperature of approximately 37-42°C. The process can be divided into two parts: reverse transcription (RT) and recombinase polymerase amplification (RPA). Initially, the linear RNA synthesized during IVT is reverse transcribed into complementary DNA which serve as template in the following RPA process (Figure 15). Subsequently, modified primers recognize its complementary sequence and insert into the double-stranded DNA template through the strand-displacement activity of the recombinase (Figure 15.①) . The single-stranded binding protein (SSB) then stabilizes the displaced DNA strand (Figure 15.②). Finally, the recombinase dissociates (Figure 15.③) to allow polymerase binding and elongation of the modified primer (Figure 15.④ and Figure 15.⑤) [12].

Figure 15. The mechanism of recombinase polymerase amplification (RPA).

PCRD: The RT-RPA products (double-stranded DNA with modified primers) serve as sample to test the functionality of PCRD.

Click to go Results G!

Reference

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[2] El-Tholoth, M., Branavan, M., Naveenathayalan, A., & Balachandran, W. (2019). Recombinase polymerase amplification-nucleic acid lateral flow immunoassays for Newcastle disease virus and infectious bronchitis virus detection. Molecular biology reports, 46(6), 6391-6397. https://doi.org/10.1007/s11033-019-05085-y

[3] Obi, P., & Chen, Y. G. (2021). The design and synthesis of circular RNAs. Methods (San Diego, Calif.), 196, 85-103. https://doi.org/10.1016/j.ymeth.2021.02.020

[4] Lee, K. H., Kim, S., & Lee, S. W. (2022). Pros and Cons of In Vitro Methods for Circular RNA Preparation. International journal of molecular sciences, 23(21), 13247. https://doi.org/10.3390/ijms232113247

[5] Lu, D., Chatterjee, S., Xiao, K., Riedel, I., Huang, C. K., Costa, A., Cushman, S., Neufeldt, D., Rode, L., Schmidt, A., Juchem, M., Leonardy, J., Büchler, G., Blume, J., Gern, O. L., Kalinke, U., Wen Tan, W. L., Foo, R., Vink, A., van Laake, L. W., … Thum, T. (2022). A circular RNA derived from the insulin receptor locus protects against doxorubicin-induced cardiotoxicity. European heart journal, 43(42), 4496-4511. https://doi.org/10.1093/eurheartj/ehac337

[6] Boss, M., & Arenz, C. (2020). A Fast and Easy Method for Specific Detection of Circular RNA by Rolling-Circle Amplification. Chembiochem: a European journal of chemical biology, 21(6), 793-796. https://doi.org/10.1002/cbic.201900514

[7] Liang, Z., Zhang, J., Wang, L., Song, S., Fan, C., & Li, G. (2007). A Centrifugation-based Method for Preparation of Gold Nanoparticles and its Application in Biodetection. International Journal of Molecular Sciences, 8(6), 526-532. https://www.mdpi.com/1422-0067/8/6/526

[8] Hwu, S., Garzuel, M., Forró, C., Ihle, S. J., Reichmuth, A. M., Kurdzesau, F., & Vörös, J. (2020). An analytical method to control the surface density and stability of DNA-gold nanoparticles for an optimized biosensor. Colloids and surfaces. B, Biointerfaces, 187, 110650. https://doi.org/10.1016/j.colsurfb.2019.110650

[9] Reddy Banda, S., Klapproth, H., Smit, N., Bednar, S., Brandstetter, T., & Rühe, J. (2022). An advanced and efficient asymmetric PCR method for microarray applications. Frontiers in bioengineering and biotechnology, 10, 1045154. https://doi.org/10.3389/fbioe.2022.1045154

[10] Boss, M., & Arenz, C. (2020). A Fast and Easy Method for Specific Detection of Circular RNA by Rolling-Circle Amplification. Chembiochem: a European journal of chemical biology, 21(6), 793-796. https://doi.org/10.1002/cbic.201900514

[11] BLobato, 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

[12] Zhang, W. S., Pan, J., Li, F., Zhu, M., Xu, M., Zhu, H., Yu, Y., & Su, G. (2021). Reverse Transcription Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a for Facile and Highly Sensitive Colorimetric SARS-CoV-2 Detection. Analytical chemistry, 93(8), 4126-4133. https://doi.org/10.1021/acs.analchem.1c00013