Ultrasensitive nucleic acid detection based on phosphorothioated hairpin-assisted isothermal amplification

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Design and working principle of the PHAmp reaction

Figure 1 illustrates the working principle of the PHAmp reaction. The key component that actuates the PHAmp reaction is the HP consisting of three functional domains: PS-DNA at the 5′ overhang, a target recognition site in the loop and stem region, and the SP region along the 3′ end. In principle, the PHAmp reaction can be divided into two reaction phases. Phase 1 accomplishes target recognition, target recycling, and production of extended HP (EP), whereas phase 2 produces an intermediate double-stranded (ds) DNA product (IP) and final dsDNA product (FP).

Figure 1
figure1

Schematic illustration of the phosphorothioated hairpin-assisted isothermal amplification (PHAmp) for detection of a target nucleic acid. This figure was created using PowerPoint Professional Plus 2016 (https://www.microsoft.com/ko-kr/microsoft-365/powerpoint).

In the absence of target DNA, the HP retains its initial structure, and the SP region is kept partially blocked by the stem-loop structure. Therefore, the PHAmp reaction does not proceed. In the presence of target DNA, however, it binds to the target recognition site within HP to open the HP, consequently promoting a foldback reaction that rearranges the SP region to form a self-priming structure at the 3′ end. The following strand displacement DNA synthesis then produces EP whose stem end is composed of a PS-DNA/DNA duplex and concomitantly recycles target nucleic acid to initiate another phase 1 reaction by binding to another free HP. The EP then enters and initiates the phase 2 reaction. Due to the reduced thermal stability of the stem end of the EP, the EP would be more readily denatured at the end under the reaction temperature (37 °C) during dsDNA breathing phenomenon, which allows the trigger to anneal to the 3′ end of EP31,32. Extension of the trigger annealed to the EP produces an IP, which also contains a PS-DNA/DNA duplex at one end. The trigger also binds to the denatured 3′ DNA of the IP in the same manner and is extended to produce an FP without any PS-DNA modification, simultaneously displacing and recycling the EP to initiate another phase 2 reaction. The combination of these two reaction phases would produce a large number of EPs and FPs, which can be monitored in real-time via duplex-specific SYBR Green I staining.

Feasibility of the PHAmp reaction

To validate the feasibility of the PHAmp reaction, we conducted the PHAmp reactions for synthetic 59-mer target ssDNA under various combinations of the reaction components and measured the time-dependent fluorescent signals produced from SYBR Green I staining for the reaction products. As presented in Fig. 2a, the rapid enhancement of the fluorescence signal was obviously observed only from the sample containing all reaction components which include HP, trigger, and DNA polymerase with target DNA (curve 2). In contrast, the fluorescence signal was negligible when the target DNA was omitted from the reaction sample (curve 3). When the trigger essential to the initiation and operation of the phase 2 reaction was excluded from the reaction sample, the fluorescent signal increased very slightly but was still quite low even in the presence of the target DNA. The slight increase in signal can be ascribed to the accumulation of EP produced by the phase 1 reaction, which is promoted only through the binding of target DNA to HP, but does not require the trigger strand. To verify that the PS modification at the 5′ overhang of HP is essential for the phase 2 reaction, a negative control HP with the same base sequence but without any PS modification at the 5′ overhang was examined for its capacity to actuate the PHAmp reaction. The negative control HP is capable of producing an EP through binding to the target DNA, but it is not able to initiate the phase 2 reaction because the stem end of the EP produced from the negative control HP without PS modification is just composed of normal DNA/DNA duplex and would not be readily denatured under the reaction temperatures. Therefore, there should be no trigger annealing site available within the EP. As expected, the fluorescence enhancement was quite negligible in this case (curve 4) and was almost the same as that seen in the absence of a trigger (curve 1). The above results confirm that the PHAmp reaction is only initiated when the target DNA is present, and the PS modification at the 5′ overhang of HP is critical to actuate the PHAmp reaction by destabilizing the PS-DNA/DNA duplexes within EP and IP and allowing the trigger to anneal to the EP and IP followed by the strand displacement DNA synthesis.

Figure 2
figure2

Feasibility of the PHAmp reaction. (a) Time-dependent fluorescence intensities produced from SYBR Green I staining during the PHAmp reaction (1: HP + Target DNA + Polymerase, 2: HP + Target DNA + Trigger + Polymerase, 3: HP + Trigger + Polymerase, and 4: Negative control HP + Target DNA + Trigger + Polymerase). The final concentrations of HP, trigger, polymerase, and target DNA are 50 nM, 1 μM, 0.125 U/μL, and 20 nM, respectively. (b) Polyacrylamide gel electrophoresis image of the PHAmp products (M1: Target DNA, M2: HP, M3: Trigger, M4: HP + Target DNA, 1: HP + Target DNA + Polymerase, 2: HP + Target DNA + Trigger + Polymerase, 3: HP + Trigger + Polymerase, and 4: Negative control HP + Target DNA + Trigger + Polymerase). In the PAGE analysis, the final concentrations of HP, trigger, polymerase, and target DNA are 500 nM, 1 μM, 0.125 U/μL, and 200 nM, respectively.

We next conducted polyacrylamide gel electrophoresis (PAGE) analysis for the products obtained from the PHAmp reactions to further support the fluorescence results. As shown in Fig. 2b, only lane 2, which contains all reaction components, showed an intense band corresponding to the FP of the PHAmp reaction. In contrast, only the applied HP band was observed in the absence of target DNA (lane 3). When the trigger was excluded or HP was replaced with a negative control HP, a strong band corresponding to the phase 1 product, EP, but no band for the phase 2 product, FP, was observed (lanes 1 and 4). These PAGE results strongly support the real-time fluorescence results.

We also performed melting curve analysis to obtain the further evidence for the formation of the reaction products during the PHAmp reaction. As shown in Figure S1, a clear peak corresponding to the melting point of FP (89.5 °C) was observed only from the sample containing all reaction components (curve 4), whereas only the melting point peak for unreacted HP (81 °C) was observed in the absence of target DNA (curve 5). A peak corresponding to the melting point of the phase 1 product, EP (88.5 °C), was also correctly observed when the trigger was excluded or HP was replaced with negative control HP (curves 3 and 6). All these results are quite consistent with the observations from the real-time fluorescence and PAGE analysis, again ensuring that the proposed PHAmp reaction operates according to the mechanism envisioned in Fig. 1.

Optimization of the PHAmp reaction

The PHAmp reaction employs an HP with a PS modification at the 5′ overhang, and the length of the PS-DNA bases within HP should greatly influence the overall performance of the PHAmp reaction because the PS modification is exclusively responsible for initiating and operating the phase 2 reaction. To examine the effect of the length of the PS-DNA bases on the melting temperature, we first conducted melting curve analysis for PS-DNA/DNA duplexes with several selected lengths, which were then compared with the corresponding DNA/DNA duplexes. As shown in Figure S2, the melting temperatures of the PS-DNA/DNA duplexes were somewhat lower than those of the DNA/DNA duplexes in all cases, due to the reduced thermal stability caused by the PS modifications. They were estimated to be all higher than 55 °C and slightly increased as the length of the PS-DNA bases increased from 13 to 20. We envisioned that the trigger strand composed of normal bases could anneal to the stem end of EP and IP by replacing the less stable PS-DNA strand during spontaneous local conformational fluctuations within dsDNA although the melting temperatures of PS-DNA/DNA duplexes are still higher than the reaction temperature (37 °C). This DNA breathing phenomenon has been very effectively utilized to achieve several key isothermal amplification methods31,33,34.

We next conducted the PHAmp reactions by employing the HPs having different numbers of PS modification at the 5′ overhang and examined the time-dependent fluorescent intensities produced from the reaction products. As a result shown in Figure S3, the HP with 15 PS modifications solely resulted in the drastic fluorescence enhancement while the other HPs did not show any significant target-specific signal. We would interpret these results as follows: The HP(13) having short 13 PS-DNA modifications should accompany the same short Trigger(13), whose annealing on either EP or IP is not sufficiently stable to allow efficient extension in the following processes. On the other hand, the HP(17) and HP(20) having longer PS-DNA modifications should lead to the more stable PS-DNA/DNA duplex at the stem end due to the increased number of hybridized bases, which would greatly reduce the probability for the trigger to anneal to the stem end of EP and IP during dsDNA breathing dynamics and initiate the phase 2 reaction. Based on these results, the HP(15) was employed as an optimal HP in all experiments of this work.

We also investigated the optimum reaction conditions, including the concentrations of HP, trigger, and polymerase as well as the reaction temperature by comparing the time-dependent fluorescence intensities of the reaction mixtures with target DNA to those without target DNA. The results showed that the following combination was optimal: 25 nM HP, 1 μM trigger, 0.125 U/μL DNA polymerase and a 37 °C incubation temperature (Figure S4, S5, S6 and S7). Thus, these conditions were used in further experiments.

Sensitivity of the PHAmp reaction

The sensitivity of the PHAmp reaction was determined by conducting the PHAmp reactions for target DNAs at a series of concentrations and measuring the real-time fluorescence signals from the reaction products. As shown in Fig. 3a, the threshold time (Tt), defined as the time when the fluorescence signal reaches the threshold intensity (1000 a.u.), decreased gradually as the target DNA concentration increased in the range from 1 fM to 1 nM. When the Tt values were plotted as a function of the logarithm (log) of the target DNA concentration (Fig. 3b), an excellent relationship (Tt =  − 15.75 log (Ctarget/M) + 190.25, R2 = 0.994) was obtained, confirming that the PHAmp reaction is quite capable of quantitatively identifying target DNA. The limit of detection (LOD = 3σ/S, where σ is the standard deviation of blank and S is the slope of the calibration curve) was estimated to be 0.29 fM, which is superior to previous alternative methods of isothermal amplification (Table S2).

Figure 3
figure3

Sensitivity of the PHAmp reaction. (a) Time-dependent fluorescence intensities (via SYBR Green I staining) during the PHAmp reaction with target DNA at various concentrations. (b) Linear relationship between Tt and the logarithm of the target DNA concentration in the range from 1fM to 1 nM, where Tt is defined as the time at which the fluorescence signal reaches the threshold intensity (1000 a.u.) and Ctarget is the target DNA concentration. The error bars represent the standard deviations of three replicate measurements.

Specificity of the PHAmp reaction

The specificity of the PHAmp reaction was assessed by employing several nonspecific DNAs, including one- (MT 1), two- (MT 2), and three-base mismatched DNA (MT 3) and non-complementary DNAs (NC1 and NC2). The NC1 has non-complementary sequence within the entire strand, whereas NC2 has random sequence only at the HP binding site. We first predicted the ΔG values for HP and its complexes with specific or nonspecific target strands by using the OligoAnalyzer tool provided by IDT (Coralville, IA, USA). The results presented in Table S3 show that the ΔG is the most negative for HP/Target complex and its negative value decreased as the number of the mismatched bases increased from one to three as expected, indicating that the incorporation of the mismatched bases to target molecules destabilizes the formation of their complexes with HP35.

The experimental data presented in Fig. 4 confirms that the Tt value from the target DNA is much smaller than those from the nonspecific targets such that the target DNA could be very reliably discriminated even from the only single base-mismatched target DNA. We might attribute this high specificity to the intrinsic mechanism of PHAmp reaction based on the stable secondary HP structure. More specifically the PHAmp reaction is not initiated only by the hybridization of target with HP but the hybridization affinity should be strong enough to fully open the hairpin structure of HP36,37. We assume that slightly weaker interaction of several base-mismatched target DNA with HP could lead to the much more intensified influence on the final signals, as evidenced in Fig. 4.

Figure 4
figure4

Specificity of the PHAmp reaction. The Tt values for target DNA and nonspecific target DNAs including several base-mismatched DNAs (MT1, MT2, and MT3) and non-complementary DNAs (NC1 and NC2) were determined and compared. Tt is defined as the time when the fluorescence signal reaches the threshold intensity (1000 a.u.).

Practical applicability of the PHAmp reaction

We verified a practical diagnostic capability of the PHAmp reaction to reliably detect relatively long target nucleic acids which were prepared by asymmetric PCR of Neisseria gonorrhoeae plasmid DNA (target DNA) and Leptospira interrogans genomic DNA (NC DNA) followed by gel-based purification of ssDNA. It should be noted that this technique was developed to identify ss target nucleic acids as manifested in Fig. 1 but we conducted asymmetric PCR just to prepare relatively long ss target nucleic acids. As presented in Fig. 5, the 221-mer long target DNA was also successfully detected, showing almost the same Tt values with that from the synthetic 59-mer target DNA whereas the NC DNA produced very negligible fluorescence signal much like the blank sample without any target. These observations verify that the PHAmp method is capable of identifying target nucleic acids without limitation for their lengths.

Figure 5
figure5

Practical utility test. Time-dependent fluorescence intensities (via SYBR Green I staining) during the PHAmp reaction in the presence of target DNA of different lengths. 221 bp DNAs were obtained from asymmetric PCR and the final concentration of the target DNAs was 20 nM.

To further evaluate the practical applicability of this strategy for complex heterogeneous biological samples, the PHAmp reaction was carried out for target DNAs in diluted human serum. As shown in Figure S9, the concentration of target DNAs spiked in the human serum also showed an excellent linear relationship with Tt values (Tt =  − 11.425 log (Ctarget/M) + 9.2451, R2 = 0.992). Based on this linear relationship, the recovery tests were performed using target DNAs at three different concentrations. Table 1 shows that target DNA concentrations were very reliably determined in human serum with high reproducibility and precision as evidenced by the recovery rates between 92.01 and 115.04%. These results suggest that the proposed method could reliably determine target nucleic acids even in complex heterogeneous biological samples containing several interfering agents38,39,40,41.

Table 1 Recovery test for synthetic 59-mer target DNA spiked into diluted human serum.

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