Funds from the UCF Office of Undergraduate Research (OUR) for AK are greatly appreciated. Work on optimization of LATE-PCR conditions for the NANOGP8 gene is supported by the Florida Cancer Innovation Fund (grant MOABC).

1. Design of the Split Light-up Aptamer Sensor (SLAS) with DAP-10-42 as a signal reporter
<ol>
	<li>Design the sequences of two unmodified DNA oligonucleotide strands constituting SLAS. Use a fragment containing nucleotides (nts) 4,6-39 of DAP-10-42 (Table 1, Figure 1A).</li>
	<li>Convert the sequence of stem 1 (nts 1-8 and 36-42) to stem 1' by removing a bulging thymine residue at position nt 5, shortening the stem to 4 bp (nts 4,6-8 and 36-39), and adding a terminal C-G bp (Figure 1A, step 1). Additionally, one of the SLAS strands contains nts 9-29 of the DAP-10-42 core sequence, while another SLAS strand contains nts 30-35 of the sequence.</li>
	<li>Extend the 3'-terminal sequence of the fragment containing nts 9-29 with d(GGTCAT), and the 5'-terminal sequence of the nts 30-35 fragment with d(ATGACC) to form a 6-bp stem 2 (Figure 1A, step 2). 
	NOTE: The base pairs T4-A39, A6-T38, C7-G37, and G8-C36 of stem 1 in DAP-10-42 are required for the light-up function of the aptamer. The sequence of stem 1' can be modified only at the terminal bp and can be a G-C, A-T, T-A base pair or extended with 1-2 base pairs as needed. The sequence of stem 2 is arbitrary and can be changed. However, it is recommended that the thermodynamic stability of stem 2 is preserved unless the assay temperature is raised above 28 °C. Shortening or otherwise destabilizing stem 2 prevents premature dye binding without a target if a high background of the SLAS-based fluorescent assay is observed. Splitting of the DAP-10-42 nucleotide sequence between nts 28-29, 27-28, or 26-27 instead of nts 29-30 is also allowed (Figure 1B).</li>
	<li>To tailor the designed SLAS to a nucleic acid target or interest, extend stem 1' with DNA sequences complementary to the target-target-binding fragments (arms) through d(TT) linkers (Figure 1A, step 3). This yields the sequences of strands SLAS-U and SLAS-S constituting the probe.</li>
	<li>Make the target-binding arm of SLAS-S complementary to a 7-10-nt fragment of the target containing the SNS site, and the target-binding arm of SLAS-U – complementary to a 15-25 nt fragment of the target adjacent to the target-binding arm of SLAS-S (Table 1). 
	NOTE: It is optional whether to connect the target-binding arm of SLAS-S to nt 4 or nt 39 of the DAP-10-42 sequence (Figure 1, arm 1 or arm 2, respectively).</li>
	<li>Assess the melting temperature (Tm) of the duplexes between the target of interest and the target-binding arms of SLAS-S and SLAS-U using the UNAFold Web Server (unafold.org)19.
	<ol>
		<li>Click on the DINAMelt tab on top of the web page; go to Applications and select Two State Melting Hybridization (Supplementary Figure 3A). On the Two-State Melting Hybridization page, enter one of the interacting sequences in the left box and another in the right box (Supplementary Figure 3B). Make sure both sequences are in 5' to 3' order. You can enter more than one pair of interacting sequences by separating them with semicolons.</li>
		<li>At the bottom of the page, indicate the assay temperature (22 °C), monovalent and divalent cation concentrations (20 mM and 25 mM, respectively) to match the assay conditions. Indicate the concentration of the interacting sequences.</li>
		<li>Press Submit (Supplementary Figure 3B). Observe the results that include the Gibbs energy change, enthalpy change, and entropy change of the binding, as well as the Tm values for the corresponding duplexes (Supplementary Figure 3B).</li>
		<li>Make sure the Tm values for the duplexes between the fully matched target (M) and the target-binding arms of SLAS-S and SLAS-U are above the assay temperature, which is 22 °C in the current protocol. Here, the Tm values were 32.2°C and 74.4 °C, respectively (Supplementary Figure 3C).</li>
		<li>Ensure the duplex between SLAS-S and a mismatched target (MM) containing an SNS has a Tm below the assay temperature. This ensures high selectivity of the probe-target recognition, preventing the AO fluorogen from signaling the presence of the targeted nucleic acid analyte if it contains an SNS in the SLAS-binding fragment (Figure 2). Here, the Tm was 21.3 °C (Supplementary Figure 3C), which was sufficient to discriminate the mismatch from the matched complex.</li>
		<li>Adjust the sequences of the target-binding arms by shortening or extending the target-complementary fragments, if needed, to meet the stability requirements indicated.</li>
	</ol>
	</li>
	<li>Obtain the designed strands SLAS-S and SLAS-U from any commercial vendor supplying custom-made DNA oligonucleotides or synthesize the DNA strands in-house using an automated oligonucleotide synthesizer.</li>
</ol>
<img alt="RNA editing diagram with sequence modification steps, showing core, stem, and target-binding arms." class="xfigimg" src="/files/ftp_upload/68483/68483fig1.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Conversion of a fluorescent light-up aptamer (FLAP) DAP-10-42 into a split light-up aptamer sensor (SLAS). (A)The secondary structure of DAP-10-4212 is predicted by a DNA folding application of the UNAFold Web Server19 at 22 °C, 20 mM Na+, 25 mM Mg2+. The aptamer features a hairpin secondary structure with a core that presumably folds into a G-quadruplex motif stabilized by stem 1. For DAP-10-42 to be converted into a SLAS, (1) stem 1 of the aptamer is shortened to 4 bp and flanked by an additional G-C bp to make stem 1'; (2) the core sequence is split between nts 29 and 30, with the 3'- and 5'-sides of the split site extended with complementary sequences d(GGTCAT) and d(ATGACC), respectively, to make stem 2; (3) stem 1' is extended with the sequences complementary to the adjoining fragments of a target of interest - target-binding arms, with a d(TT)-linker between the target-binding arms and the signal reporting domain of the probe. (B) The core can also be split between nts 28-29, 27-28, or 26-27 without significant loss of the signal and/or fluorescence fold-increase. <a href="https://www.jove.com/files/ftp_upload/68483/68483fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="SLAS-target interaction diagram showing high and no signal states with DNA complex configurations." class="xfigimg" src="/files/ftp_upload/68483/68483fig2v2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: SLAS signal generation mechanism. SLAS is composed of two strands - SLAS-U and SLAS-S - and a fluorogenic dye (e.g., Auramine O; AO). In the absence of a nucleic acid target of interest, both strands are in the dissociated state and unable to bind the dye; no signal is observed (center). In the presence of a fully matched target M, both SLAS-U and SLAS-S hybridize to the adjoining fragment of M to bring the core fragments in proximity. This allows for the formation of the dye-binding site. Binding of AO enhances its fluorescence, which is measured as a fluorescent signal readout (left). In the presence of a mismatched target MM containing a single-nucleotide substitution (SNS) in a fragment interacting with SLAS-S, only SLAS-U can hybridize to the MM target, while SLAS-S is in the dissociated state. This prevents the formation of the dye-binding site, and no signal is observed (right). <a href="https://www.jove.com/files/ftp_upload/68483/68483fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Amplification of the targeted sequence
<ol>
	<li>Amplify a fragment of the gene to be analyzed with the SLAS assay. For PCR or RPA amplification, use asymmetric conditions (e.g., with the Forward Primer added at 10-fold excess over the Reverse Primer) to generate a single-stranded amplicon. To amplify a 126 nt fragment from the NANOGP8 gene using Linear After The Exponential (LATE) PCR20, use the primer sequences listed in Table 2. Here, a single-stranded DNA template (Template NANOGP8 or Template G1423C, Table 2) corresponding to the 126-nt fragment of the gene was used. The template was custom-synthesized by Integrated DNA Technologies, Inc.
	<ol>
		<li>Prepare a 100 µL PCR reaction by mixing 10 µL of 10x Standard Taq Reaction Buffer, 2 µL of 10 mM dNTP mix (to have the final concentration of 0.2 mM), 10 µL of 10 µM Forward primer and 10 µL of 1 µM Reverse Primer (for the final concentration of 1 µM and 0.1 µM, respectively), 0.5 µL of 5U/µL Taq DNA polymerase, DNA template and nuclease-free water. For a no-template control, add water instead of the template.</li>
		<li>Use the following thermocycling conditions: initial denaturation at 95 °C for 1 min; 45 cycles of (1) denaturation at 95 °C for 15 s, (2) primer annealing at 60 °C for 30 s, (3) primer extension at 68 °C for 30 s; and final extension at 68 °C for 5 min. 
		NOTE: Transcription-based amplification techniques such as NASBA18 can be used instead of PCR or RPA, especially if an RNA template is available for amplification. NASBA generates a single-stranded RNA amplicon at a final product concentration in the micromolar range, which is high enough to be used in the downstream SLAS assay without product concentration.</li>
	</ol>
	</li>
	<li>Precipitate the amplified samples with ethanol by adding 0.1x volume of 3 M sodium acetate (pH 5.5) and 3x volume of ethanol. Incubate at -20 °C for 1 h, centrifuge at 12,000-18,000 x g for 5 min, and discard the supernatant.</li>
	<li>Wash the pellet with 70% ethanol, re-centrifuge, and discard the supernatant again. Air dry the pellet and dissolve in 10 µL of nuclease-free water to be used in the SLAS assay.</li>
</ol>
3. Fluorescence assay using SLAS
<ol>
	<li>Prepare stock solutions of AO (0.1 mM in DMSO, 10x), SLAS-S (10 µM in nuclease-free water, 10x), SLAS-U (10 µM in nuclease-free water, 10x), and 4x assay buffer (80 mM Tris-HCl, pH 7.4, 100 mM MgCl2, 80 mM KCl). 
	NOTE: Potassium ions are necessary to stabilize the presumed G-quadruplex motif of the aptamer, which is part of the dye-binding site. As little as 1 mM KCl (at 25 mM MgCl2) in 1x assay buffer is sufficient to ensure ~14-fold fluorescence enhancement in the presence of the specific target M over the blank (no-target sample; Supplementary Figure 2B). Magnesium ions stabilize the interactions of the nucleic acid strands (between SLAS-S, SLAS-U, and the target). The concentration of Mg2+ in the 1x Assay Buffer should be at least 5 mM (at 20 mM KCl; Supplementary Figure 2C,D). The stock solution of AO (~1 mM) is prepared in DMSO. Stock concentrations of SLAS-S and SLAS-U are arbitrary and can be adjusted if needed as long as the final concentrations of the strands are maintained (see the rationale for the selection of the final SLAS concentration in Discussion). 
	CAUTION: AO is harmful if swallowed, toxic in contact with skin, and suspected of causing cancer. Always wear gloves when working with AO solutions. The weighing of AO and preparing of its stock solutions should be done under the chemical hood.</li>
	<li>Prepare the master mix containing all assay components but the target. Calculate the master mix volume (Vmix) by multiplying the sample volume (60 µL) by the number of samples plus one (n+1): 
	<img alt="Mixing volume formula \(V_{mix}=(n+1)\times60\mu\text{L}\), useful in solution preparation." src="/files/ftp_upload/68483/68483eq01.jpg" style="margin: auto;" data-altupdatedat="1788304762000" data-alt="Equation 1"> 
	The number of samples n should include necessary negative and positive controls.</li>
	<li>Combine 0.25x Vmix µL of 4x Assay Buffer, 0.1x Vmix µL of 0.1 mM AO, 0.1x Vmix µL of 10 µM SLAS-S, and 0.1x Vmix µL of 10 µM SLAS-S. Add nuclease-free water to the final volume of <img alt="Mathematical expression 5/6 × Vmix; used in volume calculation formula for mixture preparation." src="/files/ftp_upload/68483/68483eq02.jpg" style="margin: auto;" data-altupdatedat="1788304762000" data-alt="Equation 2"> such that it accommodates the target volume added at step 3.4. Vortex and briefly centrifuge the master mix. Aliquot 50 µL of the master mix into n sample tubes. 
	NOTE: The sample volume can be adjusted depending on the characteristics of the instrument utilized to detect the fluorescent signal. The volume of the master mix that is aliquoted into sample tubes can be adjusted based on the target volume added at step 3.4. The volume of nuclease-free water added to make the master mix should be adjusted correspondingly. For example, if the target-containing sample is added at 10% (V/V) of the final sample volume, water should be added into the master mix up to 0.9x Vmix.</li>
	<li>Use one sample as a no-target control (blank), and one sample as a positive control. Add 10 µL of the target-containing sample to the master mix (50 µL) to make the final sample volume of 60 µL containing 10-1000 nM target. For the blank, use nuclease-free water (10 µL) instead of the target. For positive control, use a synthetic DNA oligonucleotide containing the targeted sequence (e.g., target M, Table 1). Add 10 µL of 0.6-3 µM control to make 0.1-0.5 µM final concentration. 
	NOTE: High fluorescence in this positive control ensures the assay works as expected, even if a negative signal is observed for the tested unknown samples.</li>
	<li>Mix the samples and spin down using a microcentrifuge. Incubate the samples at 22 °C for 10-60 min. The signal increases only by &lt;15% between 40 min and 60 min (Figure 3).</li>
	<li>Measure the fluorescence of the samples at 540 nm upon excitation at 475 nm using a fluorescent spectrophotometer. 
	NOTE: It is possible to use a portable fluorometer that measures fluorescence in thin-walled PCR tubes. The fluorometer needs to have excitation and emission filters suitable for detecting AO fluorescence. An example of a suitable excitation filter is a 495 nm short-pass. An example of a suitable emission filter is 510-580 nm. Alternatively, the signal can be observed visually upon excitation of AO fluorescence in the sample with a UV or Blue light source. 
	​CAUTION: If a UV lamp is used, wear UV-protective goggles.</li>
</ol>
<img alt="Kinetics graph, fluorescence increase over time, target M added; measuring F_540 RFU vs. time." class="xfigimg" src="/files/ftp_upload/68483/68483fig3.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Time-dependence of the signal generation for SLAS-29U/30S. A sample containing AO, SLAS-29U and SLAS-30S was analyzed using the kinetics mode of a fluorescent spectrophotometer (excitation at 475 nm, emission at 540 nm, 5-nm excitation/emission slits) over 60 min. At a 5 min time (indicated with an arrow), target M was added to the sample. The data is averaged from two independent trials. <a href="https://www.jove.com/files/ftp_upload/68483/68483fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors declare no conflict of interest.

In the reported protocol, the critical steps include (i) the SLAS design; (ii) assay temperature; (iii) presence of mono- and divalent metal cations (K+ and Mg2+); (iv) concentrations of SLAS strands.
SLAS design 
In designing SLAS strands, the thermodynamic stability of the complexes formed by SLAS-S and SLAS-U strands in the absence of targets, and in the presence of either specific or non-specific target (if SNS-differentiation is needed), is important to consider. Stability of the SLAS-S/U complex can be modulated by changing the sequences of stems 1&#39; and 2 (Figure 1). The nucleotide sequences of the stems should be designed to prevent association of SLAS-S and SLAS-U strands in the absence of the targeted nucleic acid sequence, thus ensuring a low background signal in the absence of the analyzed target. However, extreme destabilization of stems 1&#39; and/or 2 by shortening or changing the sequence can compromise the target-induced signal, which depends on the efficient formation of the fluorogen-binding site in the SLAS-target complex.
Another important element of the SLAS design is the target-binding arms of strands SLAS-S and SLAS-U. When SNS differentiation is needed, the target-binding arm of SLAS-S should be short enough to ensure formation of the target-strand duplex under the assay conditions (temperature and ion concentrations) only in the presence of the fully complementary target sequence. This can be assessed through the calculation of the melting temperature for the corresponding duplexes. In the reported protocol, the assay temperature is 22&#160;&#176;C. Therefore, for the duplexes formed by either the fully matched or single-base mismatched target and strand SLAS-S, the Tm values were 32.2 &#176;C (above the assay temperature) or 21.3&#160;&#176;C (below the assay temperature), respectively (Supplementary Figure 3C). This ensured high selectivity of the SLAS assay. Indeed, the target sequence MM containing a G&#62;C substitution in the fragment complementary to the target-binding arm of SLAS-S triggered a signal at the blank level (Figure 4). For the duplex between the target-binding arm of SLAS-U and the target, a Tm of 74.4&#160;&#176;C was predicted for the reported SLAS sequence (Supplementary Figure 3C). This promoted the association of SLAS with the target.
Finally, the splitting of the DAP-10-42 core sequence to distribute the signal reporting domain of the aptamer between SLAS-S and SLAS-U strands needs to be considered. The allowed splitting positions are between nts 29-30, 28-29, 27-28, or 26-27 (Figure 4, Supplementary Figure 4). SLAS-28/29 promotes high AO fluorescence in the absence of the target (Figure 4A), which results in a significant loss of the target induced fluorescence enhancement (measured as a ratio between the signal in the presence of target M and the blank - F/F0), as compared with SLAS-27/28 or SLAS-29/30 (Supplementary Figure 4A). Another potential splitting site - nts 26-27 - yields a SLAS exhibiting the second-best signal-to-blank ratio (Supplementary Figure 4A) but a somewhat low target-dependent AO fluorescence (Supplementary Figure 4B). Splitting of the DAP-10-42 nucleotide sequence between A13-G14, G14-G15, G15-G16, G18-T19, T19-G20, G22-G23, and G32-G33 (Figure 1A) results in no signal enhancement in the presence of target M (Supplementary Figure 4). This agrees with a hypothesis that the dye is sequestered at the interface between a terminal G-tetrad of a G-quadruplex motif and the last base-pair of stem 1, which is based on our preliminary data. Therefore, it can be speculated that splitting next to guanines in positions nts 14-16, 18, 20, 22, 23, 32, and 33 prevents folding of the G-quadruplex motif and, correspondingly, binding of the AO fluorogen.
The two portions of the split DAP-10-42 core can be used interchangeably with the target-binding arms of SLAS-S and SLAS-U, as the SLAS, composed of SLAS-30U and SLAS-29S, exhibits similar performance to that of the SLAS containing SLAS-29U and SLAS-30U (Figure 4B). The expected complexes with the fully matched target for both SLAS variants featuring the core split between nts 29-30 are shown in Figure 4C.
Assay temperature 
The SLAS sequences described in this protocol are designed to operate at the assay temperature of 20- 28 &#176;C, with 22 &#176;C being the optimal one. The temperature predetermines the sequences of stems 1&#39; and 2, as well as the lengths of the target-binding arms (Figure 1). Increasing the temperature above 28&#160;&#176;C decreases the maximal achievable signal for the current SLAS designs16. Even though strengthening of stem 2 can provide a way to optimize the SLAS constructs to operate at a higher temperature16, raising the assay temperature above 37-41&#160;&#176;C may not be possible due to the stability of the fluorogen-binding motif of the light-up aptamer used in the SLAS design. This limits the applications of SLAS.
Cations 
As with many hybridization probes, SLAS performance depends on the presence of mono- and/or divalent cations. Magnesium ions play a crucial role in stabilizing the hybridization process by shielding the negative charges on the DNA strands, which helps in the formation of stable duplexes23. Potassium ions, while also contributing to duplex stabilization, are required for stabilization of G-quadruplex24. The SLAS assay requires 1.25-40 mM potassium and 2.5-50 mM magnesium cations for optimal performance (Supplementary Figure 2).
SLAS strand concentration 
In the reported protocol, the two strands constituting SLAS are used at 1 &#181;M concentration each. Previously, we utilized SLAS at 0.5 &#181;M strands13. This concentration is predetermined by the affinity of the aptamer to AO (Kd=0.7 &#181;M)13. The concentration of SLAS-U can be increased to promote the formation of the fluorogen-binding complex between SLAS strands and the fully matched target. Previously, the strand was used at as high as 4 &#181;M14. The concentration of SLAS-S strand needs to be kept at 0.5-1 &#181;M to ensure a low background signal and a low signal in the presence of a mismatched target. Varying the SLAS strands&#39; concentration is a way to optimize the probe&#39;s performance.
Advantages and limitations of SLAS 
The protocol for nucleic acid analysis using SLAS described here is robust and can be used with complex samples, such as products of DNA amplification using asymmetric PCR (Figure 6) or RNA amplification using Nucleic Acid Sequence Based Amplification (NASBA)13,14. The assay can be used to quantify the amount of the amplicon in the sample using a calibration curve (Figure 3). The ability of the SLAS to quantify the amount of the NASBA-generated amplicon has been previously demonstrated13. The generated fluorescent signal can be instantaneously measured using a range of instruments - a high-performance fluorescence spectrophotometer or a portable and low-cost filter fluorometer (Figure 7A,B). Moreover, the signal can be observed visually in a near instrument-free manner, with the light source for the fluorogen&#39;s excitation as the only equipment needed (Figure 7C). The presence of a specific target can be detected in as little as 5 min, with the maximal signal achieved within 60 min (Figure 3). Importantly, the presence of a highly similar nucleic acid sequence differing in as little as a single nucleotide triggered the signal at the blank (no-target) level, thus highlighting an unprecedentedly high specificity of the multicomponent probes4,7, including SLAS11.
In the reported protocol, the sequence of DAP-10-4212 was used as a signal reporter for the designed SLAS. This FLAP exhibits promiscuity in binding different fluorogens - dapoxyl sulfonyl dyes12,13, thioflavin T13, N-methyl mesoporphyrin IX13, and a variety of aryl methane dyes including auramine O (AO) and crystal violet13. We have demonstrated previously that the analytical performance of SLAS based on DAP-10-42 is not affected much by substituting one fluorogen for another13. This opens the possibility of selecting the most suitable fluorogen for a particular DAP-10-42 SLAS application. For example, here we used AO as a fluorogen, which has a negligible fluorescence in aqueous solutions (a quantum yield of 0.0000425), while undergoing a &#62;1000-fold maximal fluorescence enhancement and ~104-fold increase in quantum yield in complex with DAP-10-4213. AO is a green emitter (&#955;em=540 nm at &#955;ex=475 nm). If a red emitter is more suitable, crystal violet can be used without changing the SLAS design13.
The sequence of DAP-10-42 core features multiple clusters of guanines, suggesting a G-quadruplex motif as part of the aptamer&#39;s functional structure. Based on our preliminary data, fluorogens are likely bound between the bottom G-tetrad of the G-quadruplex motif and the top base-pair of stem 1 (Figure 1) in a sandwich-like fashion, ensuring &#960;-&#960; continuity and a change in polarity of the dye&#39;s microenvironment characteristic for environment-sensitive dyes26. The importance of the duplex-G-quadruplex architecture for the function of DAP-10-42 as a FLAP restricts the positions of the core that can be used for splitting into SLAS. Previously, for all reported DAP-10-42-based SLAS, the core was split between nts 28-2912,13,14. Here, we demonstrated that this is not the most optimal splitting site as it yields a high no-target signal (40-90-fold increase over the signal for just AO, which is 0.10&#177;0.02 RFU), which may compromise the linear dynamic range and the limit of detection for the SLAS assays. We recommend splitting the DAP-10-42 sequence between nts 29-30 or nts 27-28, as such SLAS ensures a no-target signal only 6- or 13-fold higher than AO fluorescence, respectively.
One of the SLAS limitations is the assay temperature. The low-temperature requirement may present a challenge of pairing SLAS with one-pot nucleic acid amplification protocols, as the lowest temperature required for isothermal nucleic acid amplification techniques (e.g., RPA17 or NASBA18) is 37 &#176;C. Moreover, to be efficiently interrogated by SLAS, the amplicon needs to be single-stranded. Therefore, if RPA is used, for example, the amplification reaction should be performed under asymmetric conditions, with the primer yielding the targeted nucleic acid sequence to be used in excess over the limiting primer. Another SLAS limitation is that the limit of detection (LOD) is in the nanomolar range13,14, which hinders amplification-free applications of the assay. At the same time, the LOD is similar to that for one of the state-of-the-art hybridization probes - the MB probe3. Despite a few limitations noted for SLAS, this is a promising tool for cost-efficient nucleic acid analysis as part of molecular diagnostic and food and water safety monitoring assays, as well as a research tool in biomedical sciences and nucleic acid biotechnology.

Nucleic acid analysis using hybridization probes is routinely performed to detect and quantify specific DNA/RNA sequences1. One of the most successful hybridization probes is the Molecular Beacon (MB) probe, which is a hairpin-shaped DNA 
oligonucleotide&#160;labeled with a fluorophore and a quencher at the opposite termini2,3. It instantaneously (without the need to wash the probe-target complex from the excess of the probe) detects the presence of a nucleic acid sequence complementary to the loop fragment of the probe and is commonly used in quantitative PCR (qPCR). The need to covalently attach two dyes - the fluorophore and the quencher - to the oligonucleotide sequence of the probe increases the cost, particularly when a new MB probe targeting a new nucleic acid sequence is needed. Alternatively, the MB probe can be used as a signal reporter in a multicomponent (split) hybridization probe format, in which two unlabeled oligonucleotides interact with both the target and the MB probe reporter4. The multicomponent probe approach enhances selectivity for nucleic acid recognition and improves the probe&#39;s binding to structured targets5,6,7, This approach, nevertheless, does not eradicate the need for&#160;covalent labeling of the reporter with a&#160;fluorophore8.
Fluorescent Light-up Aptamers (FLAPs)9 are aptamers that have an affinity to low-fluorescent environment-sensitive dyes (fluorogens), where binding by FLAP enhances the fluorescence of the fluorogen manifold. Unlike fluorescently labeled probes and signal reporters, FLAPs do not require sensing elements to be covalently labeled with one or more dyes and thus offer the advantage of label-free fluorescence detection10. This is particularly important during the optimization step when dozens of nucleic acid constructs need to be tested to achieve satisfactory assay performance. Moreover, FLAP assays can be optimized by adjusting the concentration of a fluorogen and/or FLAP sensor rather than changing the sensor&#39;s design. These advantages can be significant for low-budget research and teaching laboratories. DNA FLAPs are particularly promising as signal reporters of multicomponent hybridization probes referred to as Split Light-up Aptamer Sensors (SLASs)11. The most efficient DNA FLAP reported so far is DAP-10-42, which was originally selected to enhance the fluorescence of dapoxyl sulfonyl dyes12, with a promiscuous ability to bind and enhance the fluorescence of a variety of aryl methane dyes (e.g., auramine O, AO; crystal violet, CV)13. DAP-10-42 has been converted into SLAS to be used for nucleic acid analysis13,14,15.
The goal of the method described here is to enable sequence-specific analysis of nucleic acid targets of interest using label-free SLAS strands and a fluorogenic dye as a buffer component. This protocol describes an algorithm to design SLAS based on DAP-10-42 and highlights SLAS performance for highly selective detection of nucleic acid sequences differing in as little as a single nucleotide.
The SLAS nucleic acid detection assay requires two custom-synthesized unmodified DNA oligonucleotides used at a final concentration of 0.5-1 &#181;M for maximal performance. The protocol described here utilizes a commercially available Auramine O (AO) dye as a fluorogen. The final AO concentration in the assay ranges from 1-10 &#181;M to ensure high fluorescence enhancement in the presence of a specific nucleic acid target over the blank (absence of the target; Supplementary Figure 1). The necessary components of the assay buffer are potassium and magnesium ions (Supplementary Figure 2). Potassium ions (1.25-40 mM) stabilize the proposed G-quadruplex domain of the FLAP used as a signal reporter. Magnesium ions (2.5-50 mM) reduce repulsion of negatively charged phosphate groups upon SLAS-target interactions. The temperature of the SLAS assay using the reported sequences for the signal reporting domain should not exceed 28&#160;&#176;C to avoid loss of performance16.
SLAS is compatible with single-stranded nucleic acid targets, such as DNA amplicons of asymmetric PCR or an isothermal DNA amplification reaction (e.g., recombinase polymerase amplification, RPA17), RNA products from transcription-mediated amplification (TMA) or nucleic acid sequence-based amplification (NASBA)18, and native RNA molecules such as miRNA, rRNA or ncRNA). Variations in the nucleotide sequence of the target down to a single nucleotide can be detected using the SLAS approach13,14.

<table><tbody><tr><td>Auramine O</td><td>MP Biomedicals&amp;nbsp;</td><td>195064</td><td>a fluorogenic dye used for signal reporting</td></tr><tr><td>Cary Eclipse Fluorometer</td><td>Agilent Technologies</td><td>G9800A</td><td>to measure fluorescent signal of the samples</td></tr><tr><td>Dimethy Sulfoxide</td><td>MP Biomedicals&amp;nbsp;</td><td>196055</td><td>solvent to prepare stock solutions of Auramine O</td></tr><tr><td>DNA oligonucleotides (primers, SLAS strands, synthetic template for PCR)</td><td>IDT</td><td>N/A</td><td>custome-made and desalted by the vendor, used without further purification</td></tr><tr><td>dNTP mix, 10 mM</td><td>ThermoFisher Scientific</td><td>R0191</td><td>for LATE PCR reaction</td></tr><tr><td>Magnesium Chloride</td><td>invitrogen</td><td>AM9530G</td><td>buffer component; needed for correct association of the probe&#039;s strands with the analyzed target</td></tr><tr><td>Microcentrifuge tubes (1.5-mL )</td><td>Fisherbrand</td><td>02-681-320</td><td>plastic for sample preparation</td></tr><tr><td>Micropipette (0.1-2.5 &amp;micro;L)</td><td>Eppendorph</td><td>K56877G</td><td>used for sample preparation</td></tr><tr><td>Micropipette (100-1000 &amp;micro;L)</td><td>Eppendorph</td><td>K13160F</td><td>used for sample preparation</td></tr><tr><td>Micropipette (20-200 &amp;micro;L)</td><td>Eppendorph</td><td>J05253M</td><td>used for sample preparation</td></tr><tr><td>Micropipette (2-20 &amp;micro;L)</td><td>Eppendorph</td><td>G24675G</td><td>used for sample preparation</td></tr><tr><td>Mini Centrifuge</td><td>Corning&amp;nbsp;</td><td>6770</td><td>used for spinning down the samples&amp;nbsp;</td></tr><tr><td>Nuclease-Free Water (not DEPC-Treated)</td><td>ThermoFisher Scientific</td><td>AM9932</td><td>Use for samples in PCR and SLAS assay</td></tr><tr><td>PCR tubes (0.5-mL)</td><td>invitrogen</td><td>AM9932</td><td>to measure fluorescence using Quantus fluorometer</td></tr><tr><td>Pipett tips (20-200 &amp;micro;L)</td><td>VWR</td><td>76322-150</td><td>plastic for sample preparation</td></tr><tr><td>Pipette tips (1-10 uL)</td><td>VWR</td><td>76322-528</td><td>plastic for sample preparation</td></tr><tr><td>Potassium Chloride&amp;nbsp;</td><td>Invitrogen</td><td>AM9640G</td><td>buffer component; needed for correct folding of the fluorescent light-up aptamer domain of the probe</td></tr><tr><td>Quantus Fluorometer</td><td>Promega</td><td>E6150</td><td>portable low-cost fluorometer that can be used for fluorescence measurements</td></tr><tr><td>Quartz cuvette</td><td>Starna</td><td>16.50F-Q-10/Z15</td><td>to be used for fluorescence measurement using Cary Eclipse fluorometer</td></tr><tr><td>Taq DNA Polymerase with Standard Taq Buffer</td><td>New England BioLabs</td><td>M0273S</td><td>for amplification of a gene fragment to be analyzed with the SLAS assay&amp;nbsp;</td></tr><tr><td>Tris-HCl buffer (pH 7.4)</td><td>Boston Bioproducts</td><td>R-314</td><td>buffer component</td></tr><tr><td>UV lamp</td><td>UVP</td><td>95-0016-14</td><td>to excite AO for visual signal observation</td></tr><tr><td>Vortex Mixer</td><td>VWR</td><td>10153-838</td><td>used for mixing the samples</td></tr></tbody></table>

DNA-based fluorescent light-up aptamers (FLAPs) are promising for bioanalytical assays because they provide a low-cost fluorescent signal readout without the need for labeling of nucleic acid signal reporters with fluorophores and/or quenchers, unlike conventional hybridization probes used for instantaneous nucleic acid detection. Instead, FLAPs non-covalently bind dye ligands , which exhibit intrinsically low fluorescence in aqueous solutions, but become highly emitting upon FLAP binding. This protocol describes an algorithm to design split light-up aptamer sensors (SLASs) utilizing DAP-10-42, the most efficient DNA FLAP reported thus far. When equipped with nucleic acid sequences complementary to a nucleic acid target of interest, SLAS is a promising tool for nucleic acid analysis allowing for the sequence-specific detection of nucleic acid targets with selectivity down to one nucleotide to enable analysis of single-nucleotide substitutions (SNSs). SLASs offer the advantage of a label-free fluorescence-based signal readout, which can be measured with a conventional cuvette-based fluorescent spectrophotometer, a portable fluorometer, or visually observed upon excitation with a handheld light source. The SLAS approach is beneficial for biosensing applications in disease diagnostics, environmental monitoring, and biomolecular research.

Performance of SLAS utilizing DAP-10-42 core as a signal reporter was demonstrated using a probe designed to interrogate a fragment of the NANOGP8 gene important for the progression of glioblastoma multiforme (GBM), an aggressive form of brain cancer21. This fragment was reported to contain SNSs in the DNA cargo of exosomes derived from cancer cells22. Therefore, the SLAS was tailored to the SNS site of the gene. Synthetic DNA targets M or MM mimicking the sequence of the NANOGP8 gene containing either G or C at nt 1423 (GenBank NM_001355281.2), respectively, were used to demonstrate the SLAS performance in this protocol. Target M was fully complementary to the SLAS-S strand, while target MM contained a G&gt;C mismatch with SLAS-S (Table 1). The sequence of the target-binding fragment of SLAS-U was fully complementary to both M and MM targets (Table 1).
SLAS increases the fluorescence of AO in a time-dependent manner only when a target complementary to the target-binding arms of the probe is present (Figure 3). This signal results from a tripartite complex between the target, SLAS-S and SLAS-U strands, which allows for the bridging of the DAP-10-42 fragments and formation of the fluorogen-binding site (Figure 2, left). The signal reaches a plateau after ~45-50 min incubation of the SLAS with the target. However, it is possible to observe the presence of the fully complementary target with a signal-to-blank ratio of 10 after as little as 10 min after the addition of the target (Figure 3). If the analyzed nucleic acid sequence is different from the target of interest in a single nucleotide (e.g., in the case of G&gt;C substitution in target MM as compared to target M), one of the SLAS components - SLAS-S - does not bind to the analyte, which prevents formation of the fluorogen-binding site despite SLAS-U/analyte binding (Figure 2, right). As a result, the fluorescence signal observed in the SLAS assay for target MM is at the blank level (no-target control; Figure 4). Therefore, observation of the fluorescence intensity above that of the blank for an analyzed sample can be interpreted as the presence of the target of interest (fully complementary to the target-binding arms of SLAS) in the sample.
<img alt="Gene expression analysis; RFU bar charts and DNA structure diagrams; fluorescence quantification." class="xfigimg" src="/files/ftp_upload/68483/68483fig4.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: SLAS performance. (A) Fluorescence of AO triggered by the SLAS variations targeting the same target M but having the core split at different positions (nts 27-28, nts 28-29 or nts 29-30, as indicated; Table 1). The data from three independent trials were averaged, with standard deviations shown as error bars. (B) Fluorescence of AO with SLASs with the core split between nts 29 and 30 - SLAS-29U/30S (also shown in panel (A)) and SLAS-29S/30U (Table 1). The fluorescent signal in panels (A) and (B) is measured at 540 nm upon excitation at 475 nm in the absence of targets (Blank) or in the presence of either the mismatched (SNS-containing) target MM (MM) or the fully matched target M (M). The data from three independent trials were averaged, with standard deviations shown as error bars. (C) Expected complexes between target M and the two SLASs from panel B. The sequence for the target-binding arm of SLAS-S is the same for SLAS-29U/30S and SLAS-29S/30U. The SNS site is highlighted. <a href="https://www.jove.com/files/ftp_upload/68483/68483fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The fluorescence intensity in the presence of the target in the SLAS assay depends on the concentration of the target in the sample (Figure 5). The signal increases linearly with the target concentration in the range of 0-500 nM (when SLAS-S and SLAS-U components of the assay are at 1 µM). This allows calculation of the limit of detection (LOD) and limit of quantification (LOQ) for the assay using the 3.3σ/S and 10σ/S rule, where σ is the standard deviation of the blank (no-target sample), and S is the slope of the linear dynamic range. For the SLAS-27U/28S, a LOD of 5.1 ± 0.9 nM and a LOQ of 15.3 ± 2.7 nM were obtained, which is consistent with the values obtained for SLAS previously13. The fluorescence intensity corresponding to the LOQ is less than 2-fold higher than that of the blank (F0). Therefore, the normalized fluorescence (F/F0) of 2 can be used as a threshold. The signal above the threshold indicates the presence of the targeted nucleic acid sequence in the sample.
<img alt="Linear regression graph, formula F/F₀=0.04737*[M]+1, chemistry experiment, data analysis." class="xfigimg" src="/files/ftp_upload/68483/68483fig5.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5: Dependence of SLAS signal on target concentration. Fluorescence of the probe composed of SLAS-27U and SLAS-28S was measured in the presence of varying target M concentration (0-1000 nM). Fluorescence signal (F) in the presence of the target was normalized by the blank (F0), and F/F0 was plotted as a function of the target concentration. The signal increased linearly with the target concentration in the range of 0-500 nM, with the slope (analytical sensitivity) of 0.04737 nM-1 calculated using a linear fit of the Origin software. The data is presented as the average from three independent replicates, with error bars as standard deviations. <a href="https://www.jove.com/files/ftp_upload/68483/68483fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The representative results for the analysis of samples containing the targets corresponding either to the NANOGP8 gene or its G1423C mutant obtained by LATE PCR are shown in Figure 6. Based on the emission spectra of the samples (Figure 6A), only the PCR sample 2 elicited a fluorescent signal higher than the sample containing the PCR no-template control (NC). Target M (Table 1) was used as a positive control (PC) at 0.1 µM concentration. While the normalized fluorescence of sample 2 (F/F0=6.9 ± 0.6) exceeds a threshold of 2 described above, sample 1 exhibits a F/F0 of 1.3 ± 0.2 (Figure 6B). Therefore, only sample 2 contains a sequence that is fully complementary to the SLAS target-binding arms and features a guanine at nt 1423. Based on the calibration curve (Figure 5), the concentration of the ssDNA amplicon in sample 2 can be estimated as 124 ± 13 nM. 
<img alt="Fluorescence emission spectra and bar chart of absorption results for optical analysis at 500-580 nm." class="xfigimg" src="/files/ftp_upload/68483/68483fig6.jpg" data-altupdatedat="1788304762000" data-alt="Figure 6"> 
Figure 6: Analysis of the products of LATE PCR amplification of the NANOG gene using the SLAS assay. (A) Representative emission spectra of the analyzed samples upon excitation at 475 nm. Both excitation and emission slits were 10 nm. Samples 1 and 2 contained 16.7% (v/v) of the 10-fold concentrated amplicons. NC is a negative control containing the PCR no-template control sample. Blank and PC correspond to the samples containing SLAS-27U and SLAS-28S in the absence of targets and in the presence of 0.1 µM target M, respectively. (B) Normalized fluorescence at 540 nm calculated by dividing the fluorescence of the samples (F) by that of the blank (F0). The data is the average from two replicates, with standard deviations shown as error bars. A threshold of F/F0=2 is shown with a black horizontal line to differentiate between samples containing the fully matched targeted sequence from its SNS-containing variation. <a href="https://www.jove.com/files/ftp_upload/68483/68483fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The SLAS signal can be measured using a cuvette-based high-performance benchtop fluorescence spectrophotometer or a portable filter-based fluorometer measuring the fluorescence in PCR tubes. Indeed, the observed signal pattern (Figure 7A) and signal-to-blank ratios (Figure 7B) were similar for the same samples analyzed using different instruments. For the portable filter fluorometer, the sample volume had to be increased to 100 µL (from a 60 µL volume sufficient for the benchtop spectrophotometer) due to the intrinsic instrument's restrictions. Moreover, the signal can be observed visually upon excitation of the dye's fluorescence using a UV light source (Figure 7C). However, the LOD/LOQ values for visual signal observation are likely higher than if measured with a fluorometer. Here, sample M exhibits an F/F0 of ~20 (Figure 7B).
<img alt="Fluorescence intensity analysis; bar graphs and UV tube image; Cary Eclipse vs. Quantus." class="xfigimg" src="/files/ftp_upload/68483/68483fig7.jpg" data-altupdatedat="1788304762000" data-alt="Figure 7"> 
Figure 7: Signal readout options. (A) Fluorescence of the samples containing (1) AO only (AO), (2) AO with SLAS-27U and SLAS-28S (Blank), (3) AO, SLAS-27U, SLAS-28S with the SNS-containing target MM (MM), (4) AO, SLAS-27U, SLAS-28S with the fully complementary target M (M), and AO with DAP-10-42 as a positive control (PC). Fluorescence was measured either in a 3 mm cuvette using a high-performance benchtop fluorescence spectrometer (excitation at 475 nm, emission at 540 nm, with 5-nm excitation and emission slits) or in 0.5 mL PCR tubes (100 µL) using a portable filter-based fluorometer (blue channel). The samples were prepared in triplicate, with the average values shown, and the error bars indicating the standard deviation. (B) The fluorescent signal in the presence of either fully complementary (M) or mismatched (MM) target, normalized by the fluorescence of the blank. Green and blue bars 
correspond to the data obtained using a cuvette-based fluorescence spectrometer (excitation at 475 nm, emission at 540 nm) and a portable filter-based fluorometer (blue channel; 495 nm short-pass excitation and 510-580 nm emission), respectively. (C) The signal of the samples shown in (A) is visualized upon excitation using a compact UV lamp (254 nm). The image is taken using a smartphone camera. <a href="https://www.jove.com/files/ftp_upload/68483/68483fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Sequences of oligonucleotides for the SLAS assay. Nucleotides of stem 1 and stem 2 are in blue and cyan, respectively; nucleotides of the target-binding arms and their complementary fragments of the target sequences are in green; the target-binding arm for the SLAS-30U strand is in brown; the target-binding arms of SLAS-S are underlined; single-nucleotide substitution positions and their complementary nucleotides in SLAS-S are in orange; the nucleotides of the d(TT) linkers are in grey. <a href="https://www.jove.com/files/ftp_upload/68483/Table%201_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Sequences of primers and templates used for LATE PCR. Primers and primer-binding regions are underlined. Nucleotides of the templates/amplicons interacting with the target-binding arms of SLAS-27U and SLAS-28S are in green and blue, respectively; single-nucleotide substitution positions are in orange. <a href="https://www.jove.com/files/ftp_upload/68483/Table%202_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Figure 1: Dependence of SLAS fluorescence on AO concentration. (A) Raw fluorescence of the blank (no-target) sample or the sample containing 0.5 µM target M. (B) Signal-to-blank ratio (F/F0) calculated from the data in (A). A symbol * indicates a statistically significant difference between the F/F0 values for samples containing 10 µM and 20 µM AO with p≤0.05 based on unpaired t test. The F/F0 values for the samples containing 1-10 µM AO are not statistically different. Strands SLAS-27U and SLAS-28S were tested. The data represents the average from three independent experiments, with standard deviations indicated by error bars. <a href="https://www.jove.com/files/ftp_upload/68483/S.%20Figure%201_RE.jpg" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: Dependence of SLAS fluorescence on cations' concentration. Dependence is shown on concentration of (A,B) potassium ions at 25 mM MgCl2 and (C,D) magnesium ions at 20 mM KCl in the assay buffer. (A,C) Raw fluorescence of the blank (no-target) sample or the sample containing 0.5 µM target M. (B,D) Signal-to-blank ratio (F/F0) calculated from the data in (A) and (C). Strands SLAS-27U and SLAS-28S were tested. The data represents the average from at least three independent experiments, with standard deviations indicated by error bars. <a href="https://www.jove.com/files/ftp_upload/68483/S.%20Figure%202_RE.jpg" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: Screenshots from the UNAFold Web Server to obtain Tm values. (A) Selection of Two-State Melting Hybridization application from the DINAMelt tab. (B) Entering oligonucleotide sequences interacting with each other (color coded), indicating assay conditions (temperature, ion concentrations, concentrations of the interacting oligonucleotides). The sequences of target M or MM and the target-binding arm of SLAS-28S are highlighted in yellow or green, respectively. The sequences of targets M or MM and the target-binding arm of SLAS-27U are highlighted in cyan or red, respectively. (C) Results showing the thermodynamic parameters of duplex formation. The calculated Tm values are highlighted in agreement with the colors of the interacting oligonucleotide sequences used in (B). <a href="https://www.jove.com/files/ftp_upload/68483/S.%20Figure%203_RE.jpg" target="_blank">Please click here to download this File.</a>
Supplementary Figure 4: Testing the SLAS designs with alternative split sites of the core DAP-10-42 sequences. (A) The data is presented as fluorescence of the samples containing the fully matched target M normalized by the blank signal (samples in the absence of the target). A threshold of F/F0=2 is indicated by a vertical line. (B) Raw fluorescence data for the SLAS designs highlighted in green in (A). M and Blank represent the samples in the presence and absence of target M, respectively. The data is the average from at least three independent experiments (two and six replicates were done for SLAS-13S/14U and SLAS-29S/30U, respectively), with the standard deviations indicated as error bars. <a href="https://www.jove.com/files/ftp_upload/68483/S.%20Figure%204_RE.jpg" target="_blank">Please click here to download this File.</a>

This protocol outlines the design of split hybridization probes utilizing fluorescent light-up aptamer DAP-10-42 as a signal reporter and their application for nucleic acid detection and differentiation between targets with single-nucleotide substitutions.

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Split Hybridization Probe Utilizing a DNA Fluorescent Light-up Aptamer as a Signal Reporter for Sequence-Specific Nucleic Acid Analysis

DNA-based fluorescent light-up aptamers (FLAPs) are promising for bioanalytical assays because they provide a low-cost fluorescent signal readout without ...

Amina Rhouati

Aptamers are synthetic DNA/RNA sequences selected in vitro&nbsp;for their high affinity to the target ranging from small molecules to whole cells and microorganisms. Aptamers have rivaled antibodies as promising receptors in analytical methods due to their simple and inexpensive chemical synthesis. Different reports can easily label Aptamers, including fluorophores, enzymes, and nanomaterials, allowing their use in optical-based assays and sensors. These strategies include colorimetric, fluorescent, SPR, and luminescent techniques. In addition, electrochemical transduction can also be combined with aptamers to design highly sensitive aptasensors.Moreover, aptamers can be used in label-free biosensing strategies without reporter molecules, thus providing a fast response. Given the high affinity and specificity of aptamers, these latter have shown great promise in different fields. Aptasensors can be applied to target biomarkers for accurate diagnosis, monitoring environmental pollutants, and detecting food contaminants.&nbsp;In this collection, we welcome submissions describing innovative technologies based on aptamers combined with optical or electrochemical detection, showing examples of new applications in biomedical or environmental fields.&nbsp;&nbsp;

JoVE Bioengineering merges both physical and life sciences to understand and predict biological processes. Applying physical science tools to life science questions allow for the discovery of better technologies to measure, diagnose, and clinically treat disease.

Bioengineering

Application of Aptamers in Optical and Electrochemical Sensing Platforms

Aptamers are synthetic DNA/RNA sequences selected in vitro for their high affinity to the target ranging from small molecules to whole cells and microorganisms. Curated by Amina Rhouati. Explore Bioengineering techniques on JoVE.

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Split Hybridization Probe Utilizing a DNA Fluorescent Light-up Aptamer as a Signal Reporter for Sequence-Specific Nucleic Acid Analysis