Part:BBa_K5099012

Double-stranded seesaw gate linked to fluorophore-quencher combination.

Usage and Biology

Reporter Gates

The Nucle.io project aims to provide rapid, point-of-care diagnostics that undercut current wait times for the return and analysis of laboratory test results such as blood culture (3 days) and PCR (1 day), by performing both diagnostic amplification and result computation in one reaction. This allows clinical decision making to happen on a faster timescale in emergency medical settings where time is of the essence. Sepsis is a disease responsible for 20% of global deaths. Each hour of delayed treatment leads to an 8% increase in mortality. When infection is treated early with accurate antibiotics, downstream complications such as organ failure can be prevented. The Nucleio diagnostic uses a modular approach to achieve both accuracy and speed, leveraging the CasX protein to perform mRNA-based detection or a strand displacement cascade to perform mRNA amplification. The downstream module (computation) applies the Winner-Take-All neural network (WTA NN), which is a DNA computing architecture using toehold-mediated strand displacement reactions to analyze profiles of nucleic acids developed in Neural network computation with DNA strand displacement cascades (Qian et. al, 2011).

The diagnostic results of these modules, which allows the identification of infection-causing bacterial species and associated antimicrobial resistance markers, is reported as a level of fluorescence that increases over time. As such, a general fluorescent reporter for the diagnostic is developed which triggers upon the terminal strand displacement reaction. This reporter part can also be adapted to report the performance of the CasX and SDR amplifier modules through the use of a DNA translator gate.

Reporter Gate Design

This reporter gate consists of a simple 5nt toehold domain with a 15nt branch migration domain. This part is created from two annealed ssDNA strands, Rep[X]-t and Rep[X]-b.

To create the part, a fluorophore-quencher (FQ) pair was covalently linked 5’ and 3’ to the top and bottom oligonucleotides respectively. This linkage is on the 5’ and 3’ non-sticky ends. In McGill iGEM’s design, Rep-t-F, Rep-b-Q are annealed in a 1:1 stoichiometric ratio.

A custom synthesis of fluorophore-linked oligonucleotides was performed by Jathavan Asohan at Sleiman Lab, McGill Department of Chemistry.

To anneal oligonucleotides, both Rep[x]-t and Rep[x]-b strands are HPLC purified and diluted to a concentration of 100uM. 4.5uL volumes of top strand and bottom strand are added with 1uL of 10xMg1xTE added, and strands are heated to 95ºC and allowed to cool 1ºC/min. Reaction volumes can be scaled up as necessary. No downstream purification was done.

All fluorophore displacements are done at a 200nm concentration in 12.5mM Mg+ ion concentration.

Reaction and Characterization

We document the timescale on which this strand displacement reaction occurs (see above) as well as the fluorescence standard curve for a given 200nm stock of fluorophore. Here, the strand that displaces the incumbent strand (fluorophore) is referred to as the invading strand, shown as Y[x]. Shown is the background level of fluorescence for a quenched complex, while the level of fluorescence for a single strand displacement reaction (200nm Y[x] strand) is shown. This reaction occurs so quickly that the reaction is nearly complete in the time between adding the input strand and the start of the plate reader program (<15s), adding a small cutoff in the beginning of our graphs.

Fluorescence over concentration over time. Each timescale is shown as its own dotted line. Y strand here is the invading strand, or the trigger for the fluorescence reaction. Reaction is already partially triggered at t=0 due to the above quirk.

UNQF: Unquenched fluorophore

QF: Quenched fluorophore (baseline fluorescence level)

QF+Y: Fluorophore + Trigger strand

Standard curves showing fluorescence by concentration of trigger strand are shown below.

Fluorophore: Cy5.5 Phosphoramidite Quencher: BBQ650 CPG

A custom synthesis of fluorophore-linked oligonucleotides was performed by Jathavan Asohan at Sleiman Lab, McGill Department of Chemistry.

To anneal oligonucleotides, both Rep[x]-t and Rep[x]-b strands are HPLC purified and diluted to a concentration of 100uM. 4.5uL volumes of top strand and bottom strand are added with 1uL of 10xMg1xTE added, and strands are heated to 95ºC and allowed to cool 1ºC/min. Reaction volumes can be scaled up as necessary. No downstream purification was done.

Fluorophores were excited at 660nm and emission was at 700nm. All fluorophore displacements are done at a 200nm concentration in 12.5mM Mg+ ion concentration.

Reaction and Characterization

We document the timescale on which this strand displacement reaction occurs (see above) as well as the fluorescence standard curve for a given 200nm stock of fluorophore. Here, the strand that displaces the incumbent strand (fluorophore) is referred to as the invading strand, shown as Y[x]. Shown is the background level of fluorescence for a quenched complex, while the level of fluorescence for a single strand displacement reaction (200nm Y[x] strand) is shown. This reaction occurs so quickly that the reaction is nearly complete in the time between adding the input strand and the start of the plate reader program (<15s), adding a small cutoff in the beginning of our graphs.

Fluorescence over concentration over time. Each timescale is shown as its own dotted line. Y strand here is the invading strand, or the trigger for the fluorescence reaction. Reaction is already partially triggered at t=0 due to the above quirk.

Footnote 1: In the Qian WTA design (1), the quencher (Q) is attached to the top strand as it does not contain a toehold. The fluorophore (F) is attached to the bottom strand. This design allows for Rep[1]-t to be annealed in excess concentration (Rep[x]-t:Rep[x]-b stoichiometry 1.2:1) without creating leak effects in any subsequent strand displacement cascade.