Team:USTC/Design

Design

Aptamer  

    Aptamers are oligonucleotide that can bind to specific target molecules. The aptamer we choose is a single-stranded DNA fragment with affinity to tau protein^[1]. Comparing with antibodies, aptamers can better identify the corresponding target molecule^[2]. In addition, aptamer is cheaper and easier to modify. For these considerations, we chose the aptamer. However, not all the aptamers can meet the requirements of our experiment with the affinity of tau protein, so we need to screen aptamers to select aptamers that can bind to tau protein stronger.

    At present, the commonly used method of aptamer screening is SELEX (Systematic evolution of ligands by exponential enrichment). Although new methods such as Non-SELEX^[3]have appeared in recent years, SELEX is still the most mature screening method at present. However, it still takes a long time for SELEX to screen the aptamer for iGEM competition. Therefore, we plan to use the aptamer sequence of tau screened by predecessors. We choose tau441^[4] with its affinitive aptamer^[3]. If the affinity of these aptamers fails to meet the requirements of our project, further screening will be carried out on the basis of these existing sequences of aptamers. After screening, the sequence with the strongest affinity to tau protein will be obtained (Fig 1).

Fig 1A: SELEX

Figure adapted from: Source

Fig 1B: Testing of affinity of aptamer and tau-protein

Design of template chain  

    We designed a molecular switch with two strands——NA chain (none aptamer chain) and AP chain (the chain without aptamer). The aptamer can bind to the free region of NA chain and is not connected to AP chain by phosphodiester bond. As the figure 2 shows, the binding site of primer 1 and 2 are nonexistent if the aptamer exists.

    In the absence of aptamer, DNA polymerase will bind to the 3’ end of AP chain so the whole AP chain can be synthesized using NA chain as template. The binding site of primer 2 appears, thus the complete NA strand can also be obtained. RPA reaction can start as the complete template chains occur.

    When the aptamer is present, the aptamer will prevent the DNA polymerase from prolonging AP chain. As a result, AP strand cannot be completed and the binding site of primer 2 will not appear. RPA reaction is repressed, which will be manifested in significant decrease in fluorescence signal intensity. When the tau protein exists, tau protein will bind to the aptamer and the hydrogen bond between aptamer and chain NA breaks. The DNA polymerase can bind to chain AP and the RPA reaction can continue. We will use qPCR instrument to achieve the detection of fluorescence signal intensity.

Fig 2 design of template chain

Asymmetric PCR  

    As the template chain requires two incomplete complementary single chains, we need to obtain NA and AP ssDNA respectively using the asymmetric PCR. According to the literature, single strands can be obtained by adding two primers with different proportions^[5]. In symmetric, we need to add the same proportion of forward and backward primers to get dsDNA. If the amount of one primer is larger than the other, we can get single strands under the action of DNA polymerase. For example, if we want to get NA ssDNA, we need to add more forward primers. It is recorded that the brightest single strand can be obtained when the primer ratio is 20:1. Base on it, we designed a conditional verification experiment for asymmetric PCR. We changed primer ratios and temperature conditions respectively to perform asymmetric PCR under the same other conditions. As a result, we can find the most appropriate condition and continue to perform subsequent asymmetric PCR under this condition. The product of asymmetric PCR is a mixture of dsDNA and ssDNA, thus we need to isolate and purify the single strand if we want to get ssDNA.

RPA   

    It is necessary to amplify DNA for nucleic acid detection. In PCR, the control of temperature is indispensable. High temperature denaturation is required in PCR. Under high temperature conditions, hydrogen bonds break, aptamer is directly separated from the template chain, and polymerase can directly complement the chain, unable to prevent amplification. Besides, PCR need to be completed by instruments in the laboratory and it will take a long time. However, RPA can run with little equipment in a short time^[6]. We hope our device can be easy as possible. That’s why we choose RPA.

    Recombinase polymerase amplification (RPA) is a single tube, isothermal alternative to the polymerase chain reaction (PCR)，which can be done without complex installation. In RPA, at room temperature, the recombinant enzyme and primers can form the protein-single-stranded nucleotide complex Rec-ssDNA. With the help of the helper protein and single-stranded binding protein SSB, Rec-ssDNA can bind to its complementary segments. After the target region complementary to the primers is found, the Rec/ssDNA of the complex is disintegrated, and the polymerase also binds to the 3 'end of the primers to start the chain extension. This process is rapidly and efficiently cycled, so as to complete the ultra-fast amplification of the target fragment (Fig 3).

Fig 3 RPA

Probe  

    We designed a probe with a fluorophore and a quencher. There is a THF residue between the fluorophore and the quencher. Due to the presence of quenched groups, the probe could not display fluorescence signal. Only when the probe and its corresponding strand combine and form a double strand, exo enzyme will recognize THF residue and cut it. (Fig 4)

    Fluorescence signal will appear in the system as the fluorophore and the quencher separate^[7] . In order to facilitate, the sequence of the probe was designed to be consistent with the primers. Only after the amount of double stranded DNA is high enough, the probe will combine.

Fig 4 probe

Signal conversion  

    The template chains, primers and probe were put into the reaction system, mixed and put into the fluorescence detection device, and the fluorescence value of the system was measured every 20s to make the image of fluorescence intensity changing with time. Only when the template chains exist but the aptamer does not exist, can the fluorescence signal be detected. When both template chains and aptamer exist, but tau protein does not, the fluorescence signal should be weak and the fluorescence intensity should be significantly different from that without aptamer. The ideal result of our diognosis is that the fluorescence intensity is positively correlated with the concentration of tau protein when the template chains, aptamer and tau protein all exist.

    Without doubt we need to do parameter optimization if possible. We hope to reflect the different concentration of tau protein in the system according to the different fluorescence intensity curve. This requires adjusting parameters such as temperature, reaction time, template and primer concentration to find the most appropriate range to achieve the best result.

    The reaction condition of RPA is between 35 ° C and 42 ° C. We need to select different temperature to react and find the optimal reaction temperature interval of RPA, and we can conduct more experiments to find a more refined temperature range. The reaction time of RPA also had a great influence on the results. Too short a time would lead to insufficient amplified numbers and weak fluorescence signals. Too long a time would lead to too many amplified numbers, so the final difference of fluorescence signals will be not significant, which could not achieve the effect of distinguishing tau protein concentration. Therefore, it is necessary to find the time that can distinguish different concentrations of tau protein according to the experimental results. Obviously, the concentration of each reactant in the reaction system will greatly affect the experimental results, so we need to find the best combination of reactant concentration.

Laboratory Test Methods  

    Based on the experimental results, the following is our summary of the laboratory test method for tau protein content in blood^[8]. It is divided into three steps: blood sample separation, reaction and final signal display.

    Since the presence of blood cells may have adverse effects on our detection (many relevant literatures believe that the presence of plasma proteins will not affect the detection of tau protein, but the impact on the presence of blood cells is rarely mentioned, and all experiments are conducted using plasma or serum), blood samples should be separated before testing samples. Then, the primers and templates needed for subsequent RPA amplification and the lyophilized powder of corresponding enzymes were added to the separated plasma and reacted for a period of time. Finally, a quantitative real-time PCR was used to measure the fluorescence signal of the sample after reaction, and the intensity of the fluorescence signal was accurately converted to the concentration of tau protein by the corresponding relationship between the fluorescence signal obtained in the previous experiment and the concentration of tau protein.

Reference  

    [1]: Krylova, S.M., et al., Tau protein binds single-stranded DNA sequence specifically - the proof obtained in vitro with non-equilibrium capillary electrophoresis of equilibrium mixtures. Febs Letters, 2005. 579(6): p. 1371-1375.
    [2]: Shui, B., et al., Biosensors for Alzheimer's disease biomarker detection: A review. Biochimie, 2018. 147: p. 13-24.
    [3]: Lisi, S., et al., Non-SELEX isolation of DNA aptamers for the homogeneous-phase fluorescence anisotropy sensing of tau Proteins. Analytica Chimica Acta, 2018. 1038: p. 173-181.
    [4]: Teng, I.T., et al., Identification and Characterization of DNA Aptamers Specific for Phosphorylation Epitopes of Tau Protein. Journal of the American Chemical Society, 2018. 140(43): p. 14314-14323.
    [5]: Marimuthu, C., et al., Single-stranded DNA (ssDNA) production in DNA aptamer generation. Analyst, 2012. 137(6): p. 1307-1315.
    [6]: Stringer, O.W., et al., TwistAmp (R) Liquid: a versatile amplification method to replace PCR. Nature Methods, 2018. 15(5): p. I-III.
    [7]: Piepenburg, O., et al., DNA detection using recombination proteins. Plos Biology, 2006. 4(7): p. 1115-1121.
    [8]: Jacobs, K.R., et al., Correlation between plasma and CSF concentrations of kynurenine pathway metabolites in Alzheimer's disease and relationship to amyloid-beta and tau. Neurobiology of Aging, 2019. 80: p. 11-20.