HCR


Overview

Hybridization Chain Reaction (HCR) is a method to amplify double-stranded DNA containing many nicks in an enzyme- free manner using microRNA or single-stranded DNA as input. It requires three different templates with a hairpin structure and reacts at 37 ℃. We performed experiments based on existing methods 1.
For more information about the principle of HCR, see Proposed Implementation
For more information about the actual experimental procedure of HCR, see Wet Experiments_HCR

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1. Agarose Electrophoresis

Purpose:

We attempted to observe the amplified product by agarose gel electrophoresis.

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Consideration:

Agarose gel electrophoresis showed low resolution and blurred peaks.

2. Capillary Electrophoresis

Purpose:

Because the peak was blurred in 1., we attempted to observe the amplified product using a more sophisticated capillary electrophoresis system. The concentration of target miRNA or ssDNA corresponding to that was used as the variable.

Result:
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Consideration:

The elongated peaks were observed only at 1 µM for miRNA and at 1 µM and 100 nM for ssDNA. The paper reported that the dynamic detection range of CRISPR-HCR is 1 fM-100 nM and that of HCR is 10 pM-100 nM, but this could not be reproduced at all.

3. Optimization

Purpose:

Because the expected LoD could not be achieved in 2., changes were made to the experimental conditions.

  • Annealing may make the hairpin structure too stable → Changed whether annealing was performed or not in the experiment
  • The incubation time may not be long enough → Changed the incubation time
  • The reaction between miRNA and H1 may be too slow in the first step → Changed the H1 concentration

Result:
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  • Without annealing, the amplified product was confirmed even in the negative control.On the other hand, without annealing, the amplified product was obviously detected around 52 bp. Therefore, annealing operation was thought to be essential.

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  • The results were similar for all incubation times after 10 min, indicating that an incubation time of 10 min is sufficient when the target ssDNA is 1 μM.

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  • The H1 concentration → When peak was observed, there was a tendency for lower H1 concentrations to produce fewer amplification products. For more detailed study, the concentration corresponding to Cas target was calculated from the molecular weight and concentration values of the peak.

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The paper connects the products of HCR to Cas. Therefore, the concentration of target ssDNA/miRNA and the concentration of Cas target in the amplified product are expected to be correlated. It is expected that long amplified products contain more Cas targets and short amplified products contain fewer Cas targets.

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Consideration:

Since one Cas target is contained per H2 or H3, one Cas target is contained per 60 bp of amplified product. 1 bp is approximately 330 Da, so this formula can be used to calculate the Cas target concentrations, taking into account all Cas targets contained in the various peaks.

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4. Peak Attribution

Purpose:

For the results in 2., the peaks obtained by capillary electrophoresis were attributed to the assumed amplification product.

Notes:

Capillary electrophoresis outputs the “molecular weight" and “concentration" of the peaks.Of these, the “molecular weight" was calculated by applying the molecular weight of the peak to the width of the upper and lower ladder of known molecular weight. If the peak is dsDNA, this calculation method can be used, but the amplified product of HCR is something like dsDNA with many nicks, and a discrepancy between the “molecular weight" calculated by the machine and the actual molecular weight is expected to occur.

Therefore, we avoided this problem by applying an amplified product with a value close to the “molecular weight" of the peak during attribution. Fortunately, the attribution was not difficult because, in principle, there are only a few amplification products with molecular weights close to the molecular weight of the HCR.

Result:
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T: Target, H1: Hairpin1, H2: Hairpin2, H3: Hairpin3, e.g. T-H1-H2 represents an amplicon which consists of T, H1, H2.

Consideration:

The results of the attribution showed that the amplification products are oligomers of n=10 at the longest. This indicates that the amplification efficiency of HCR is inadequate. The MultiNA we used has a lower detection limit of 0.2 ng/µL for DNA and a quantification range of 0.5-50 ng/µL 2. Therefore, the lack of detection when the target was at low concentrations could be due to the fact that it was below the detection limit of MultiNA. However, based on the attribution of this peak, only n=10 oligomers at most were produced, and it would be difficult to amplify a concentration of PAM sufficient to activate Cas from a target of a few fM.

References


  1. Jia, H.-Y., Zhao, H.-L., Wang, T., Chen, P.-R., Yin, B.-C., & Ye, B.-C. (2022). A programmable and sensitive CRISPR/Cas12a-based MicroRNA detection platform combined with hybridization chain reaction. Biosensors and Bioelectronics, 211, 114382. https://doi.org/https://doi.org/10.1016/j.bios.2022.114382

  2. Shimadzu Corporation. (n.d.). Microchip Electrophoresis System for DNA/RNA Analysis MCE-202 MultiNA Instruction Manual. http://mor.niboch.nsc.ru/public/MBA_Course/References/Electrophoresis/
    devices/Shimadzu%20MultiNA%20manual.292-28464D_SWmanualMCE-202(E).pdf