Cheng-Han Yang

Discussion

RCA feasibility and conditions

• For RCA, two enzymes are needed: a ligase and a polymerase. Phi polymerase works best around 30 °C, but may still function at 25 °C, though with longer reaction times.
• Typical lab protocols use 2h ligation and 2h polymerization at 30 °C for single molecule detection. For our diagnostic needs, shorter times might be sufficient.
• The main challenge is finding compatible buffers for ligation, polymerization, and possibly RPA. Testing “one-pot” reactions (RPA + ligation) was suggested to evaluate feasibility.

Detection methods and signal output

• Cheng-Han pointed out that colorimetric or fluorescent outputs could be explored.
• He proposed alternatives to fluorescence, such as quenchers or FRET. Quenchers may be simpler than FRET since they don’t require specific wavelengths and could even work under sunlight.
• He emphasized that enzymatic methods can be unstable over time, so non-enzymatic quencher approaches could be more robust.

Probe design and specificity

• Hybridization depends strongly on probe length. Around 20 nucleotides should be stable at room temperature.
• Too short → risk of nonspecific binding.
• Longer or multiple recognition sites improve specificity.
• He recommended running BLAST searches against human and pathogen genomes to avoid cross-reactivity.

Main Takeaways

• RCA is possible but requires two enzymes and compatible buffers, which complicates the workflow.
• Phi polymerase is the most promising option for room temperature RCA.
• Quencher-based detection could be a practical alternative to enzyme-heavy approaches.
• Probe design is crucial, ~20 nt probes, tested against genomes, can optimize specificity.

Unanticipated Insights and New Questions raised

• Cheng-Han suggested new methods we had not considered, such as FRET and quencher systems.
• He highlighted that enzymatic systems degrade over time, raising concerns about durability in our test format.

New questions included considerations about assay design, such as whether simpler, non-enzymatic approaches (e.g., quenchers) should be prioritized over RCA to improve robustness, and which probe length and design parameters best balance sensitivity and specificity.

Integration

We implemented his advice by:

• Re-evaluating RCA as a potential detection method and considering its complexity compared to alternatives.
• Researching quencher-based detection approaches as a simpler, more robust option.
• Performing BLAST checks for probe sequences to ensure specificity.
• Deciding to focus on DNA hybridization and CRISPR-Cas as primary detection methods, while keeping RCA as a theoretical alternative.

Discussion

DNA sequence design

• He recommended starting with 18-24 bp DNA probes, and also testing LNA-modified probes for higher stability.
• Suggested ordering 3-4 different lengths (15 bp, 20 bp, 25 bp, 30 bp) and one modified probe (e.g. 15 bp DNA + 5 bp LNA).
• Begin with single binding (pathogen + nanoparticle probe) and optimize length before designing multiple hybridizations.

Visualizing hybridization and proving specificity

• Fluorescent labeling (e.g., Cy3 or Cy5) can confirm hybridization. Place dyes at opposite probe ends (5’ and 3’) to avoid interference.
• Verification methods: gel electrophoresis, comparing complexes at expected lengths.
• Signal thresholds: DNA >3 nM, LNA >1 nM, ideally >12.5 nM for visibility.

Practical considerations

• DNA concentration should be measured with a Nanodrop before and after RPA.
• Discovered that since we work with dsDNA pathogens, we only need one primer (forward or reverse) in experiments.
• Anchoring: Mobile probes can be dried onto conjugate pads; immobilized probes can be linked via streptavidin-biotin.

Main Takeaways

• Gold-nanoparticle hybridization is technically feasible and not too complex.
• Different probe lengths and LNA modifications should be tested to optimize stability.
• Fluorescent dyes and gels are key to proving correct binding.
• For dsDNA pathogens, only one primer direction is needed.

Unanticipated Insights and New Questions

• Important realization: our pathogens are dsDNA viruses, so primer use must be adapted.
• New question: What exact probe length and LNA-DNA ratio will work best in practice?

Integration

We implemented his advice by:

• Planning to order different probe lengths (with and without LNA).
• Including fluorescent dye labeling to visualize hybridization.
• Adapting primer use to forward/reverse only for dsDNA pathogens.

Discussion

Asymmetrical RPA design

• A 0:1 ratio is not possible because it would make the reaction linear. Ratios like 1:20 still allow for an exponential phase before becoming linear.
• Suggested testing with 10,000x diluted template, to simulate real conditions.
• Gel bands may appear faint due to low product amounts and single-stranded DNA. Solution: purify products, measure with NanoDrop, and load the full concentration onto a 1.5% gel.
• Caution: avoid running out of dNTPs to prevent over-amplification.
• If gels don’t work: use probes or check with PCR optimization tools.

Isothermal RPA design

• Check literature for reaction kinetics: secondary structures may slow down RPA.
• Proposed using a master mix, changing only the incubation temperature, to ensure consistency across experiments.

Prototype development

• Strongly recommended buying the Milenia HybriDetect kit to quickly generate a prototype.
• Warned that developing our own strip would require extensive optimization and time.
• With HybriDetect strips, multiplexing may be possible by combining strips.

Pathogen choice for validation

• Suggested using HPV16 L1 as a proxy but also testing with Syphilis, since no point-of-care test exists yet.
• Recommended using templates with longer sequences for easier visualization.

Other suggestions

• Always use fresh aliquots to avoid degradation.

Main Takeaways

• Clearer understanding of how to design asymmetrical and isothermal RPA assays.
• Strong suggestion to buy the HybriDetect kit to build a prototype.
• Good pathogen testing strategy: start with HPV16 L1 and Syphilis.

Unanticipated Insights and New Questions

• New insight: multiplexing could be possible with HybriDetect strips.
• Question: What exact probe design will be needed for multiplexing?

Integration

We implemented his advice by

• Planning to use HybriDetect strips for a faster prototype.
• Designing new asymmetrical RPA experiments with the correct ratios.
• Considering Syphilis as a key pathogen target for validation.