Team:Worldshaper-Shanghai/Design
Design
How to detect KLK3 and PCA3 in urine
Our solution：Collect RNA from urine→Get total cDNA by reverse transcription→cDNA Amplification → Fluorescent indication by toehold switch system
Step1: Collecting urine total RNA and converting into cDNA by reverse transcription.
Step2: PCR reaction to amplify the cDNA fragment of PCA3 and KLK3 by using their gene specific primers (T7 promoter and trigger are added on primers) .
Step 3: The amplified products were transcribed and translated with toehold switch system in vitro. The success of the reaction can be judged by the red fluorescence after the reaction. Generally speaking, our project uses fluorescence to determine whether the subject has prostate cancer: if there is red fluorescence, the subject has prostate cancer; otherwise, the subject is free from prostate cancer.
Primer design:
After we have decided what genes to look for in the case of a prostate tumor, we used various online tools to design primers for our two genes, PCA3 (BBa_K3577002) and KLK3 (BBa_K3577003) . Our first step was to look up the sequence of our targeted genes. We searched PCA3 and KLK3 on the UCSC Genome Browser（http://genome.ucsc.edu/）, a genome browser hosted by UCSC which offers access to genome sequence data. When determining which specific transcript of the gene to use, we chose the transcript with the most amount of exons (Figure 1). We then copied the sequence of one particular exon shared by multiple transcripts of the gene.
Figure 1: Results after searching gene names (PCA3 as an example). The bottommost transcript is the one we eventually chose.
After obtaining the gene sequence, we pasted it on Stanford’s Primerize (https://primerize.stanford.edu/), a web server for primer design, to yield sequences of primers. To test which primer could be added to the PCR and help to duplicate our target DNA most effectively, we use the following procedures to find which primer is the most effective to combine with our target DNA (Figure 2).
Figure 2: Primer design results on Primerize (PCA3 as an example).
Finally, we obtained three pairs of primers for each gene, and provided the sequences to a biotech company for primer synthesis. All pairs of primers of PCA3 and KLK3 will be verified by PCR ( Polymerase Chain Reaction) before we start following research, the best one pair for each gene will be used in further amplification.
cDNA Amplification:
PCR :
In order for the PCR product to be directly available for use in the in vitro transcription/translation process, we have chosen to add a T7 promoter sequence to the 5’end of forward primers and add a trigger DNA sequence to the 5’ end of reverse primers (Figure 3).
·The T7 promoter serves to provide a binding location for the RNA polymerase complex, The T7 promoter sequence is as follows:
5′ -TAATACGACTCACTATAGGG- 3′
Where the underlined G is where transcription starts. We have also followed the well-established practice to add two guanines (G) directly after the T7 sequence so as to increase PCR yields.
·The trigger sequence is as follows:
5′ - GTTTGAATGAATTGTAGGCTTGTTATAGTTATGTTT- 3′
The underlined part is the trigger binding sequence, the 5’ GTT and 3’ TTT will play their respective roles after the PCR products are transcribed into RNA: the 5’ GTT acts as the flexible end of RNA; the 3’ TTT is a universal sequence that helps ribosome binds to RBS and start translation after the stem loop of toehold switch is opened.
Figure 3 : cDNA Amplification
RPA (future plan):
We also plan to try Recombinase Polymerase Amplification (RPA) after verifying the technical route we described above for the reason of improving the detection efficiency and convenience. RPA, compared with PCR, can complete amplification in a shorter time and only needs 37 ℃ of heating, which makes the amplification reaction can be carried out without PCR equipment.
Toehold switch:
Toehold switch is a special RNA hairpin structure, which contains trigger strand, ribosome binding site, translation start codon and a report gene. Our toehold switch sequence (BBa_K3577001) is based on an article called Toehold Switches: De-Novo-Designed Regulators of Gene Expression[1](Green A, et al., 2014) and the mCherry is added to the downstream of the RNA hairpin structure (Figure 4).
Figure 4: The design of toehold switch
Toehold switch repress translation through it’s hairpin structure, which hind the RBS and start codon (AUG). The trigger sequence, a RNA sequence that complete reverse complementary with trigger strand of Toehold Switch, is a key to start translation. When the two linear RNA are complementary, the stem-loop will open, the ribosome will bind to the RBS, recognize the start codon and start translation (Figure 5 ).
Figure 5 : The activation principle of toehold switch
A.The original structure of toehold switch;
B. The trigger combines with toehold switch
C. When the hairpin structure of toehold switch opens, the ribosome binds to RBS
D. The protein is obtained by the work of ribosomes
Our haripin structure of toehold switch is as follows:
Figure 6 : The haripin structure of toehold switch
From the first red part to the first green part is the reverse complementary sequence with trigger. The dark blue 9 bases are the stem bottom sequence, the light green 6 bases are the stem top sequence that near the loop, the orange part is the loop where the yellow background is the RBS ribosome binding site (RBS ) sequence, We focus on modifying the 7 bases (CAGAAAC, orange color) before RBS, which make RBS more easily exposed and improve translation efficiency. The last black sequence is the linker, which helps to indicate the correct folding of the protein.
After in vitro transcription, the biomarker RNA will also be connected with trigger. the product with trigger can open the stem-loop structure and form a red fluorescent protein, which is the visible observation result (Figure 6 ).
After completing the design of toehold switch, we contructed T7 promoter and toehold switch as well as mCherry into pSB1C3 backbone (BBa_K3577000,Figure 7 ), which could be used as materials for in vitro transcription / translation.
Figure 7 : The pSB1C3 with T7 and toehold-mCherry
References：
[1] Green A , Silver P , Collins J , et al. Toehold switches: de-novo-designed regulators of gene expression.[J]. Cell, 2014, 159(4):925-939.
Figure 2: Primer design results on Primerize (PCA3 as an example).
Finally, we obtained three pairs of primers for each gene, and provided the sequences to a biotech company for primer synthesis. All pairs of primers of PCA3 and KLK3 will be verified by PCR ( Polymerase Chain Reaction) before we start following research, the best one pair for each gene will be used in further amplification.
cDNA Amplification:
PCR :
In order for the PCR product to be directly available for use in the in vitro transcription/translation process, we have chosen to add a T7 promoter sequence to the 5’end of forward primers and add a trigger DNA sequence to the 5’ end of reverse primers (Figure 3).
·The T7 promoter serves to provide a binding location for the RNA polymerase complex, The T7 promoter sequence is as follows:
5′ -TAATACGACTCACTATAGGG- 3′
Where the underlined G is where transcription starts. We have also followed the well-established practice to add two guanines (G) directly after the T7 sequence so as to increase PCR yields.
·The trigger sequence is as follows:
5′ - GTTTGAATGAATTGTAGGCTTGTTATAGTTATGTTT- 3′
The underlined part is the trigger binding sequence, the 5’ GTT and 3’ TTT will play their respective roles after the PCR products are transcribed into RNA: the 5’ GTT acts as the flexible end of RNA; the 3’ TTT is a universal sequence that helps ribosome binds to RBS and start translation after the stem loop of toehold switch is opened.
Figure 3 : cDNA Amplification
RPA (future plan):
We also plan to try Recombinase Polymerase Amplification (RPA) after verifying the technical route we described above for the reason of improving the detection efficiency and convenience. RPA, compared with PCR, can complete amplification in a shorter time and only needs 37 ℃ of heating, which makes the amplification reaction can be carried out without PCR equipment.
Toehold switch:
Toehold switch is a special RNA hairpin structure, which contains trigger strand, ribosome binding site, translation start codon and a report gene. Our toehold switch sequence (BBa_K3577001) is based on an article called Toehold Switches: De-Novo-Designed Regulators of Gene Expression[1](Green A, et al., 2014) and the mCherry is added to the downstream of the RNA hairpin structure (Figure 4).
Figure 4: The design of toehold switch
Toehold switch repress translation through it’s hairpin structure, which hind the RBS and start codon (AUG). The trigger sequence, a RNA sequence that complete reverse complementary with trigger strand of Toehold Switch, is a key to start translation. When the two linear RNA are complementary, the stem-loop will open, the ribosome will bind to the RBS, recognize the start codon and start translation (Figure 5 ).
Figure 5 : The activation principle of toehold switch
A.The original structure of toehold switch;
B. The trigger combines with toehold switch
C. When the hairpin structure of toehold switch opens, the ribosome binds to RBS
D. The protein is obtained by the work of ribosomes
Our haripin structure of toehold switch is as follows:
Figure 6 : The haripin structure of toehold switch
From the first red part to the first green part is the reverse complementary sequence with trigger. The dark blue 9 bases are the stem bottom sequence, the light green 6 bases are the stem top sequence that near the loop, the orange part is the loop where the yellow background is the RBS ribosome binding site (RBS ) sequence, We focus on modifying the 7 bases (CAGAAAC, orange color) before RBS, which make RBS more easily exposed and improve translation efficiency. The last black sequence is the linker, which helps to indicate the correct folding of the protein.
After in vitro transcription, the biomarker RNA will also be connected with trigger. the product with trigger can open the stem-loop structure and form a red fluorescent protein, which is the visible observation result (Figure 6 ).
After completing the design of toehold switch, we contructed T7 promoter and toehold switch as well as mCherry into pSB1C3 backbone (BBa_K3577000,Figure 7 ), which could be used as materials for in vitro transcription / translation.
Figure 7 : The pSB1C3 with T7 and toehold-mCherry
References：
[1] Green A , Silver P , Collins J , et al. Toehold switches: de-novo-designed regulators of gene expression.[J]. Cell, 2014, 159(4):925-939.
Worldshaper-Shanghai 2020
New non-invasive technique for early stage prostate cancer diagnosis