Exact tailoring of an ATP controlled streptavidin binding aptamer

Abstract

By replacing a stem of a streptavidin binding aptamer with a split ATP binding aptamer, we tailored a fused aptamer. Its binding ability to streptavidin could be controlled through the allosteric change induced by ATP binding. This aptamer engineering strategy could be used to construct other aptamer switches with desired functions.

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Aptamers have attracted intense research efforts in recent years because of their potential analytical and biomedical applications.^1,2 As molecular recognition ligands, aptamers have many advantages over antibodies.^3–5 One of the attractive advantages is the ease of aptamer synthesis and modification, which makes the molecular engineering of aptamers much easier than antibodies. Many engineering aptamers with smart properties and functions have been reported,⁶ such as aptamer based molecular devices,^7,8 aptasensors,^9–11 aptamer based therapeutic agents^12,13 and aptamer functionalized materials for programmable release of proteins or cells.^14,15 By linking a nucleic acid strand to an aptamer, some controllable allosteric aptamers have been engineered, in which the linked nucleic acid strand include another aptamer,^11,16–18 a complementary strand^19,20 or a ribozyme/DNAzyme.²¹ The functions of the engineering allosteric aptamers can be controlled through an allosteric change driven by a specific operator strand or target of integrated aptamer.^11,22–24 Aptamer based molecular switches are also found in nature, i.e. riboswitches (RNA switches) which are gene control elements that comprise a natural aptamer domain for ligand binding, and an expression platform that transmits the ligand-binding state of the aptamer domain through a conformational change.²⁵

ATP binding aptamer was selected by Szostak et al.²⁶ in 1995. It has been well characterized^27,28 and has been used to construct various sensors for ATP detection.^11,29,30 A fascinating property is that this aptamer can be split into two subunits, and the separated subunits are still able to bind to ATP through self-assembly.^26,31 In previous work, we have reported that many streptavidin binding aptamers possess a common binding motif. This binding motif is a conservative bulged hairpin structure, in which only several nucleotides in the loop and bulge area are critical for binding while other nucleotides are variable.³² This property of the streptavidin binding aptamers provides many choices to engineer this aptamer. Through substituting two canonical base pairs of an optimized streptavidin binding aptamer, St-2-1, with T–T mismatched base pairs, we have obtained a new aptamer that can be controlled by Hg²⁺ through the formation of T–Hg–T metal-base pairs.³³ By linking aptamer St-2-1 and a thrombin binding aptamer with a short DNA linker, we have designed a thrombin triggered aptamer for simultaneous separation and detection of thrombin. Chang et al.³⁴ has also designed an interferon-gamma triggered aptamer by the same strategy and realized the interferon-gamma detection with a very high sensitivity by surface plasmon resonance technique. In these engineered aptamers, the component aptamers are not or only slightly changed.

In this paper we report the construction of a fused allosteric aptamer (FAA) by replacing the stem I of St-2-1 with a split ATP binding aptamer. FAA was exactly tailored to have the streptavidin binding capacity only in the presence of ATP. The application of this FAA for ATP detection was also investigated.

The raw materials for the construction of FAAs are an optimized streptavidin binding aptamer (St-2-1) and an ATP binding aptamer. As shown in Fig. 1, St-2-1 contains a loop, a bulge and two stems, in which stem I and stem II only maintain the loop-bulge binding structure and the base pairs are changeable³² (Fig. 1). ATP binding aptamer can be split into two subunits that form heteromeric double strands upon the binding of ATP.²⁹ So if using the split ATP binding aptamer to replace stem I of St-2-1, the fused aptamer, FAA-1 (Fig. 1a) may bind streptavidin in the presence of ATP. In order to test this idea, we used streptavidin coated beads to capture the fused aptamer in the presence and absence of ATP. The captured DNA was detected by SYBR-green I staining. SYBR-green I is a popular dye, which preferentially binds to double-stranded DNA and results in great fluorescence enhancement. As shown in Fig. 2a, in the absence of ATP, the fluorescence intensity of the captured FAA-1 was very weak, indicating the low binding of FAA-1 on beads. However, in the presence of ATP, the fluorescence intensity of the captured FAA-1 was 4.7-fold higher than that in the absence of ATP, suggesting that FAA-1 was captured by the beads. These results suggest that this idea can work.

Fig. 1 (a) The design of fused allosteric aptamers (FAA). St-2-1: an optimized streptavidin binding aptamer; ABA: ATP binding aptamer. (b) The operation principle of FAA. FAA can not bind to streptavidin in the absence of ATP. The addition of ATP induces the allosteric change of FAA and results in the binding of FAA to streptavidin. The ATP bound FAA can be captured by streptavidin coated beads and stained by SYBR-green I.

Fig. 2 (a) Binding of FAAs (0.5 μM) on streptavidin coated beads in the absence (Control) and presence of ATP (1 mM). (b) CD spectra of FAA-2 (4 μM) in the absence and presence of ATP (0.5 mM). (c) Binding of FAA-2 (0.5 μM) on streptavidin coated beads in the presence of 1 mM ATP, GTP, CTP or UTP.

In order to enhance the binding ability of the fused aptamer, FAA-1 was further tailored by adding two base-pairs (T–A, A–T) at the end of FAA-1, and changing a mismatched T–T base pair to a T–A base pair at the head (the split point) of ATP binding aptamer (Fig. 1a). This modification may increase the stability of the formed heteromeric double-strand in the presence of ATP. As we expected, the new aptamer, FAA-2 strongly bound to streptavidin coated beads in the presence of ATP, while the binding was still very weak in the absence of ATP. The fluorescence intensity of the captured FAA-2 in the presence of ATP was 10.7-fold higher than that in the absence of ATP (Fig. 2a), indicating that FAA-2 is a well-tailored aptamer. The structure prediction³⁵ showed that FAA-2 formed a new secondary structure that did not contain the conservative bulged hairpin structure for streptavidin binding (Fig. 1b). The conformational transition of the FAA-2 was confirmed by circular dichroism (CD) spectroscopy. In the absence of ATP, FAA-2 adopts a conformation of B-DNA duplex with a broad positive band with maximum at 275 nm and a negative maximum at 247 nm, respectively. After the addition of 0.5 mM ATP, the broad positive band decreased and the CD spectrum exhibited a positive peak at 280 nm and a negative peak at 260 nm (Fig. 2b). This CD signal agreed well with the reported CD spectra of ATP–aptamer complex,^26,31,36 suggesting the conformational transition of FAA-2 resulted from the ATP binding. Therefore, the potential response mechanism of FAA-2 is shown in Fig. 1b: without ATP, FAA-2 could not form the streptavidin binding structure resulting in the loss of binding ability; the addition of ATP induces the two subunits of ATP binding aptamer to form a heteromeric double strand, and subsequently promotes the rest of FAA-2 to form the bulged hairpin structure and restores the binding ability to streptavidin. Compared with other allosteric aptamers constructed by combining two aptamers with a linker sequence, this fused aptamer is shorter and looks more integrated. The apparent dissociation constant (K_d) of the FAA-2 in the presence of 2 mM ATP was 78 ± 43 nM (Fig. S1†), which is approximately two fold higher than the K_d of original aptamer St-2-1 (40 ± 18 nM).³² This K_d value of the fused aptamer is still in the nanomolar range, suggesting that the displacement of the stem of St-2-1 with ATP bound aptamer did not much reduced the aptamer affinity.

The selectivity of FAA-2 to ATP was tested by measuring the fluorescence intensity of FAA-2 captured in the presence of ATP, cytidine triphosphate (CTP), guanosine triphosphate (GTP) and uridine triphosphate (UTP) respectively. As shown in Fig. 2c, FAA-2 only responded to ATP but not to GTP, UTP and CTP, indicating that FAA-2 has similar selectivity with the original ATP binding aptamer.

In order to test whether FAA-2 would work in the biological sample, we performed the same experiment in 30% RPMI 1640 medium, 30% RPMI 1640 medium with 10% fetal calf serum and 30% human plasma, respectively (Fig. S2†). In the absence of ATP, the streptavidin beads could not capture FAA-2 from these matrixes suggesting that the components in serum or cell culture medium did not cause the response of FAA-2. In the presence of ATP, the fluorescence intensities of captured FAA-2 from these matrixes were greatly increased, and respectively reached 97%, 94%, 87% of that from Tris–HCl buffer, suggesting that the components in serum or cell culture medium did not significantly interfere with the binding of FAA-2 to ATP and streptavidin. These results suggest that FAA-2 may be used in complex biological environment.

In order to test the feasibility of using FAA-2 for ATP detection, we used streptavidin coated beads to capture FAA-2 in the presence of different concentration of ATP. After washing, the beads were transferred to 50 μL centrifuge tubes respectively and stained by adding 10 μL of 1 × SYBR green I solution. As shown in Fig. 3a, the fluorescence of the captured FAA-2 was observed to enhance with the increase of ATP under UV light, as low as 50 μM ATP could be distinguished by bare eyes. Then the fluorescence intensity of these samples was measured after diluted with 90 μL of Tris–HCl buffer. The concentration–response curve to ATP (0 to 5 mM) is shown in Fig. 3b, a linear range was obtained in the range from 0 to 1000 μM (R² = 0.9825) (Fig. 3b inset). The limit of the detection was estimated to be 40 μM according to the 3σ criteria. This detection limit is comparable to some of previously reported aptamer-based ATP assays.^37,38 The concentration-dependent manner suggests that FAA-2 can be acted as a recognition element for the design of ATP sensor. In current method, the complex of aptamer and ATP was captured by streptavidin coated beads, and then indicated by SYBR green I. FAA-2 did not need to be labeled with any reporter.

Fig. 3 (a) The fluorescence under UV light of FAA-2 captured by streptavidin coated beads in the presence of different concentrations ATP (0–5 mM). (b) Plot of the fluorescence intensity versus ATP concentrations. Inset: calibration curve for ATP detection (0–1 mM).

The sensitivity of this method is not high enough, which may due to the lower affinity of the fused ATP binding moiety and the nonspecific binding by the streptavidin coated beads. The K_d of the ATP binding aptamer is 6 μM,²⁶ molecular engineering on this aptamer (such as splitting or introducing complementary sequences) would largely decrease its affinity, and decrease the sensitivity of the designed aptasensors. Through introducing a signal amplification process, ATP aptasensor may have much better sensitivity. The reported detection limits of various optical ATP aptamer sensors are in the range of 0.25 to 200 μM.^29,37–40 Combining this fused aptamer (FAA-2) with other sensing techniques such as fluorescence anisotropy, quartz crystal microbalance, surface plasmon resonance or electrochemistry may be more suitable for the direct detection of ATP with high sensitivity.

In previous study, we have reported that the bound St-2-1 and its derivatives can be easily eluted off from streptavidin coated beads by biotin, Tris–HCl buffer (pH 9.0) or 1 mM NaOH.^16,32 Elution with Tris–HCl buffer (pH 9.0) or 1 mM NaOH does not affect the binding capacity of streptavidin; therefore the streptavidin immobilized support can be reused.¹⁶ In this experiment, we observed that 10% DMSO solution could completely elute off the bound FAA-2 and then streptavidin-coated beads could be easily regenerated by washing with Tris–HCl buffer, allowing its reuse up to 5 times without significantly losing in affinity to FAA-2 in the presence of ATP (Fig. S3†). Since streptavidin and streptavidin based materials are widely used and readily available, this ATP controlled streptavidin binding aptamer holds good potential for applications in the design of molecular devices, DNA computers and so on.

With the above results, we have demonstrated that the tailored aptamer, FAA-2 has ATP controlled binding activity to streptavidin. It further confirms the variable of the stem region of the streptavidin binding aptamer. The main function of the stem region is to maintain the binding structure of streptavidin binding aptamer,³² therefore displacing this stem with a stable double-strand structure would not significantly affect the binding ability to streptavidin. In this case, the displaced sequences (subunits of ATP binding aptamer) can't form stable double-strand structure to maintain the binding structure for streptavidin in the absence of ATP. Only in the presence of ATP, the displaced sequences form a stable heteromeric double strand together with ATP and induce the formation of the binding structure for streptavidin. Therefore, ATP acts as a control element of the tailored aptamer. Many reported aptamers have the stem-loop binding motif, the stem region of some of them has been reported changeable to some extent.^19,41–44 Some other aptamers (such as cocaine binding aptamer, D-vasopressin binding aptamer, thrombin binding aptamer et al.) have also been reported could be split into two to three fragments, which could self-assemble into complex in the presence of its target.^31,45–47 Therefore, this strategy may extend to construct other aptamers with controllable function. As the targets of aptamers are ranging from small molecules, peptide, protein, even to cell and tissue,^48–50 it can be expected that the engineering aptamers will be endowed with various desired functions along with more and more aptamers being generated, such as mimicking a biological response process.

Conclusions

In summary, we have tailored a fused allosteric aptamer by replacing the stem of streptavidin binding aptamer with the split ATP binding aptamer. The fused aptamer does not bind to streptavidin in the absence of ATP. Only ATP can trigger the allosteric change of the fused aptamer and result in the binding to streptavidin. Using streptavidin coated beads to capture the complex of aptamer and ATP, and using SYBR-green I to indicate the captured aptamer, we have demonstrated that the fused aptamer exhibited linear response to ATP. The detection limit is 40 μM using fluorescence measurement. With more and more aptamers against various targets are generated and well characterized, this aptamer fusing strategy could potentially be used to construct new aptamers with desired functions.

Acknowledgements

The authors acknowledge the financial support from Grant 973 Program (2011CB935800 and 2013CB933700) and NSF of China (21275149, 21205124 and 21321003).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra00714j

‡ These authors contributed equally to this work.

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