Characterization

Overview

Lambert iGEM utilized padlock probes coupled with the rolling circle approaches to detect various microRNAs (miRNA) that are upregulated in correlation to coronary artery disease (CAD) (Fichtlscherer et. al, 2010). These mechanisms require fluorescent reporter mechanisms to quantify the initial miRNA concentration. Rolling circle amplification (RCA) used linear DNA probes, while rolling circle transcription (RCT) used the RNA aptamer Broccoli (see RCA and RCT). To supplement RCA’s results regarding the correlation of rolling circle product (RCP) with fluorescence, Lambert iGEM characterized linear DNA probes. After failed attempts at cloning, Lambert iGEM decided to continue characterization of various Spinach aptamers during 2023.

Linear DNA Probes

General Mechanism of Linear DNA Probes

Linear DNA probes as a reporter mechanism utilize both a fluorophore (donor) and a quencher (acceptor) probe. Quenchers are molecules that absorb energy from a fluorophore and re-emit a large portion of that energy as either heat or visible light (Ogawa et al., 2009). When a fluorophore and quencher are far apart, the quencher is unable to absorb the fluorophore's emission, allowing for fluorescence detection. When a fluorophore and quencher are in proximity, the quencher absorbs the energy released by the fluorophore in a fluorescence resonance energy transfer (FRET) (see Fig. 1). FRET is a distance-dependent process, where non-radiative energy gets transferred from the excited fluorophore to the quencher (Sekar & Periasamy, 2003). Following light absorption, the acceptor absorbs or "quenches" the donor's fluorescence emission energy, which is then emitted at the acceptor's emission wavelength. If the donor substance is solely present, this process produces a final fluorescence emission at a wavelength that is noticeably longer than would have been predicted.

Figure 1. Diagram showing FRET between the fluorophore and quencher.

Linear DNA Probes in CADlock

Lambert iGEM selected fluorophore and quencher-tagged linear DNA probes as one of the reporter mechanisms for rolling circle amplification (RCA) in detecting microRNA (miRNA) (see RCA). RCA produces a rolling circle product (RCP), which contains a complementary sequence to the fluorophore and the quencher. Once both the quencher-tagged and fluorophore-tagged linear probes bind to the RCP, the quencher absorbs the energy of the fluorophore, effectively diminishing the fluorescent signal (see Fig. 2). This fluorescence “shut-off” occurs due to the FRET reaction (Zhou et al., 2015). The decrease in fluorescence in the solution can be correlated using a specific concentration of miRNA through further characterization.

Figure 2. Production of repeated linear DNA probes binding sites by RCA in the presence of target miRNA.

Part Design

Basic parts, BBa_K4245130 and BBa_K4245132, are linear DNA probes designed by researchers of Key Lab at Shaanxi Normal University (Zhou et. al, 2014). BBa_K4245130 is a fluorophore molecule labeled with a 6-caryboxy-uroescein (FAM) at the 5’ end. BBa_K4245132 is a quencher molecule labeled with a Black Hole Quencher (BHQ1) at the 3’ end.

As shown in Figure 2, the fluorophore and quencher bind to the rcp, aligning the probes head-to-head. This proximity allows the quencher to suppress the natural fluorescence emitted by the fluorophore, resulting in no fluorescence output.

Experimental Design

1. Add the following to an amber microcentrifuge tube to make FAM-Probe Mastermix:
• # of reactions x 1.6 μL= μL Volume of 1 mM FAM Probe
• # of reactions x 29 μL = μL Volume of TE Buffer (pH 7.5 - 8)
2. Add the following to a PCR tube:
• 29.4 μL Mastermix
• 1.6 μL of 1 mM BHQ-1 Probe
• 1 μL of diluted complement DNA solution (0, 0.1, 1, 10, 100 μM)
3. Vortex several seconds and spin down tubes.
4. Place tubes in thermocycler at 41°C for 1 minute.
5. Place tubes in thermocycler at 37°C for 1 minute.
6. Pipette all 32 μL of solution into a 384 well plate to measure fluorescence at excitation wavelength of 480 nm and emission intensity at 518 nm using a plate reader.

Characterization Results

Lambert iGEM collected data for basic parts BBa_K4245130 and BBa_K4245132 using the Characterization Protocol (found above for reference). There is a negative logarithmic correlation between the complement concentrations ranging from 0.1-100 mM and the relative fluorescence units (RFU) (see Fig. 3). The 0 mM complement concentration outputs less RFU than 0.1 mM, which does not align with the model. However, the large error bars at 0 mM suggests that there was some degree of significant error. Thus, this data point is insignificant and further trials should be performed to achieve more accurate results. Moreover, the data from 0.1-100 mM closely parallels the predictive ordinary differential equation (ODE) model (see Fig. 4) correlating miRNA to RFU (see Modeling). The complement concentration in the characterization curve mimics RCP. Therefore, the overall data collected depicts an accurate relationship between the complement concentration and RFU.

Figure 3. Characterization curve for parts BBa_K4245130 and BBa_K4245132 showing a negative logarithmic relationship between RFU and complement concentrations ranging from 0.1-100 μM. Note: 0-0.1 μM shows positive relationship, but large error bars at 0 μM suggest this was due to faulty pipetting.

Figure 4. Deterministic ODE Model Simulation of RFU output dependent on concentration of RCP.

Aptamers

General Mechanism of Aptamers

Aptamers are short synthetic DNA or RNA sequences that utilize a three-dimensional shape to bind to specific targets, such as small molecules, amino acids, proteins, or whole cells (Ni et al., 2020). Fluorescent light-up aptamers (FLAPs) are RNA aptamers used for RNA biological studies such as cellular imaging. Both DNA aptamers and RNA FLAPs bind to and enhance the fluorescent output of specific flourogens (Pothoulakis et al., 2013). RNA FLAPs can also be encoded in DNA and transcribed, which parallels the utilization of green fluorescent protein (GFP) in biosensors. However, FLAPs can be more effective than GFP in biosensing as they bind to a fluorophore after transcription (RNA), while GFP requires additional translation for expression (Filonov et al., 2014).

Originally generated by the Jaffrey Lab at Cornell University, the Spinach aptamer (BBa_K734002) is a FLAP that binds to 3’5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), a small dye derived from the GFP fluorophore (Paige et al., 2011). The aptamer and DFHBI bind together to produce green fluorescence, which has roughly 50% of the fluorescence intensity of enhanced GFP (see Fig. 5) (Neubacher & Hennig, 2018). Similar to other FLAPs, Spinach is expressed within a transfer RNA (tRNA) scaffold, which shields the RNA from misfolding and degradation (Paige et al., 2011).

Figure 5. DFHBI and Spinach aptamer binding to form RNA-fluorophore complex.

Aptamers in CADlock

For the 2022 project, Lambert iGEM selected aptamers as one of the reporter mechanisms for rolling circle transcription (RCT) and rolling circle amplification (RCA) in detecting microRNA (miRNA) (see RCA and RCT). The team chose aptamers over other commonly used reporters due to their short length, as padlocks are more effective at lengths less than 150 nucleotides (nt) (Li et al., 2009). Lambert iGEM decided to utilize the RNA aptamer Broccoli and the DNA aptamer Lettuce for these processes but also attempted to document the similarly-behaving Spinach aptamer for further research and contribution purposes. All three aptamers bind to DFHBI; however, Broccoli and Lettuce also bind to DFHBI-1T (an improved version of DFHBI with higher photostability). RCA produces a middle sequence to which reporters bind, while RCT produces aptamers that bind with the dye after amplification or transcription eliminating the need for translation or other processing steps (see Fig. 6) (Neubacher & Hennig, 2018).

Figure 6. The transcription of repeated aptamer sequences by RCA and RCT in the presence of target miRNA and fluorescence with the addition of DFHBI-1T.

Part Design

Spinach

BBa_K4245001 is a composite part that produces the fluorescent RNA aptamer Spinach. BBa_K4245001 consists of an inducible promoter (BBa_R0010) repressed by the LacI gene, a Spinach Aptamer with stabilizing tRNA scaffolds (BBa_K734002) designed by Austin Texas iGEM 2012, and a T7 terminator (BBa_K731721) (see Fig. 7).

Figure 7. Diagram of the BBa_K4245001 construct.

The LacI protein represses the inducible promoter, which stops downstream transcription of the Spinach aptamer. When IPTG is present, LacI is inhibited, allowing for the transcription of the aptamer. Once DFHBI binds to the aptamer, the RNA-fluorophore complex produces a quantifiable green fluorescence.

iSpinach

iSpinach is a mutant of Spinach developed by researchers at the University of Strasbourg. Compared to Spinach, iSpinach is less salt-sensitive, has higher thermal stability, and produces more fluorescence (Autour et. al, 2016). To compare the function and efficacy of iSpinach to Spinach, Lambert iGEM created the composite part BBa_K4245002.

BBa_K4245002 consists of an inducible promoter (BBa_R0010) repressed by the LacI gene, the iSpinach aptamer (BBa_K3380150) designed by Edinburgh iGEM 2020 with stabilizing tRNA scaffolds (BBa_K4245004 and BBa_K4245005), and a T7 terminator (BBa_K731721) (see Fig. 8). BBa_K4245002 works identically to BBa_K4245001 (see Spinach), the only difference is the aptamer produced. This disparity allows accurate comparison of the basic aptamer parts.

Figure 8. Diagram of the BBa_K4245002 construct.

iSpinach-D5-G30-A32

As an improvement to the existing parts BBa_K3380150 and BBa_K734002, Lambert iGEM contributed part BBa_K4245000, the iSpinach-D5-G30-A32 aptamer. iSpinach-D5-G30-A32 is a co-crystallized, re-engineered version of iSpinach developed by researchers at the University of Strasbourg. They first re-engineered the original Spinach aptamer to enhance fluorescence production and promote intermolecular interactions during crystallization. However, further research identified that with few mutations in the basal stem and UNCG loop, iSpinach-D5-G30-A32 optimizes iSpinach’s production and crystallization, improving its folding capacity (Millan et. al, 2017). To compare the function and efficacy of iSpinach-D5-G30-A32 to iSpinach and Spinach, Lambert iGEM created the composite part BBa_K4245003.

BBa_K4245003 consists of an inducible promoter (BBa_R0010) repressed by the LacI gene, the iSpinach-D5-G30-A32 aptamer (BBa_K4245000) with stabilizing tRNA scaffolds (BBa_K4245004 and BBa_K4245005), and a T7 terminator (BBa_K731721) (see Fig. 9). BBa_K4245003 works identically to BBa_K4245001 and BBa_K4245002 (see iSpinach), the only difference is the aptamer produced. This disparity allows accurate comparison of the basic aptamer parts.

Figure 9. Diagram of the BBa_K4245003 construct

Experimental Design

1. Grow biosensor cells in liquid culture tubes with 5 mL LB and 5 μL antibiotic for 24 hours shaking at 170 RPM at 37°C.
2. Add 5 μL of culture cells into Erlenmeyer flasks with 50 mL LB and 50 µL antibiotic and grow for an additional 24 hours shaking at 170 RPM at 37°C.
3. Ensure that the cells are at an OD600 value between 0.4 and 0.8 using a spectrophotometer.
   • If the OD600 value is less than 0.4, grow cells for longer, checking the value every 20-30 minutes.
   • If cells have grown too much, dilute using LB until the concentration matches the desired OD600 value.
4. Add 4.5 mL of the cell culture into 15 mL liquid culture tubes.
5. Add 500 µL of diluted IPTG solution (0, 1, 10, 100 µM).
6. Grow biosensor cells for an additional 2-4 hours shaking at 170 RPM at 37°C.
7. Add DFHBI to a final concentration of 200 µM.
8. Pipette 150 µL of the grown biosensor cells into a well plate to measure cell density (OD600) and fluorescence at excitation wavelength of 492 nm and emission intensity at 501 nm using a plate reader.

Future Plans

Lambert iGEM was unable to achieve successful cloning of the Spinach, iSpinach, and iSpinach-D5-G30-A32 constructs this year (see Characterization Notebook). However, the team will continue cloning and begin characterization of this part in the upcoming year.

References