Melting Curve Analysis of Aptachains: Adenosine Detection with Internal Calibration

Abstract

Small molecules are ubiquitous in nature and their detection is relevant in various domains. However, due to their size, sensitive and selective probes are difficult to select and the detection methods are generally indirect. In this study, we introduced the use of melting curve analysis of aptachains based on split-aptamers for the detection of adenosine. Aptamers, short oligonucleotides, are known to be particularly efficient probes compared to antibodies thanks to their advantageous probe/target size ratio. Aptachains are formed from dimers with dangling ends followed by the split-aptamer binding triggered by the presence of the target. The high melting temperature of the dimers served as a calibration for the detection/quantification of the target based on the height and/or temperature shift of the aptachain melting peak.

Keywords: aptachain self-assembly; calibration/normalization; melting temperature; small molecule detection; split-aptamers.

Scheme 1

Split aptamer sequence designs. The dimers are composed of two sequences, SAX-YA and SAX-YB sorted by the number of base pairs hybridized on the left (X) and right (Y) side of the adenosine pocket. SA stands for split aptamer while the final letters A and B are Zip sequences at the 3′ end. Zip sequences A and B are complementary to each other in order to ensure dimer formation at high temperature. The aptachains formed at lower temperature are driven by the hybridization of the split aptamers (split-aptamer bridge) and possibly enhanced by the presence of adenosine (see Scheme 2). SA3-5A/B sequences present a non-complementary sequence GAG (yellow). The full aptamer Apta6 with six hybridizing base pairs at the end of the hairpin was also considered as a reference.

Scheme 2

Illustration of (A) the aptachain phase diagram with the corresponding melting temperatures and (B) the self-assembly stages. The different stages of the aptachain formation are illustrated as a function of the temperature and the adenosine concentration (A). Starting from high temperature (region 1), the dimers form due to the hybridization of the Zip sequences (region 2) at T_m(dimers). By further decreasing the temperature, aptachains form due to the split-aptamer sequences hybridization independently of the adenosine concentration (region 3). At low adenosine concentration and by further lowering the temperature, adenosine binds to the aptamer pocket (melting temperature: dashed red line leading to region 4). At a high concentration of adenosine, its binding to the aptachains enhances its stability. Consequently, the aptachains form at higher temperatures (melting temperature: full line in red), which are adenosine concentration dependent.

Figure 1

Melting curves (normalized first derivative of the UV absorbance) for the different sequence designs (a) SA3-5A/B, (b) SA5-5A/B, (c) SA3-6A/B, (d) SA5-6A/B, (e) Apta6, and (f) SA5-8A/B) with 100 µM adenosine (full line) and without (dotted line) in the solution. Peaks relate to aptamer bridges and aptachain formation (first peak) and dimers formation through Zip hybridization (second peak), respectively. The second peak maximum has been set to one for normalization. The concentrations of the DNA strands were set to 0.9 µM in all experiments. The full aptamer Apta6 only showed one peak corresponding to the formation of the aptamer bridge or hairpin configuration in this case and its maximum value was normalized to one.

Figure 2

Melting curves for the dimer design SA3-6A/B with an increasing concentration of adenosine (c = 0, 5, 10, 20, 50, and 100 µM) and for 100 µM of guanosine to assess selectivity. Oligonucleotide sequences concentrations were set at 0.9 µM.

Figure 3

(Left): First peak height maximum for SA3-6A/B dimer and various adenosine concentrations (c = 0, 5, 10, 20, 50, and 100 µM). The Langmuir fit leads to K_D = 19 µM. Error bars are smaller than the square symbols. (Right): Melting temperature of the aptachains as a function of adenosine concentration. The horizontal band represents the melting temperature without adenosine with its error bar. The red line is a logarithmic fit of the last three points. The oligonucleotides concentration in all the samples was set to 0.9 µM.

Figure 4

Melting curves normalized by the height of the dimer peak as a function of the temperature difference, T_m(dimer) − T, for three buffer conditions (0.5× green, 1× blue and 1.5× red) with 100 µM adenosine (full line) and without adenosine (dashed line). Note that due to the temperature difference expressed as T_m(dimer) − T, the aptamer bridge melting peaks are on the right side of the figure while the reference dimer melting peaks are centered at 0 °C.

Figure 5

Melting curves with 100 µM adenosine in the solution for various oligonucleotide concentrations (0.3, 0.6, 0.9, and 1.2 µM) without (Left) and with (Right) normalization.