Characterizing Aptamers with Reconfigurable Chiral Plasmonic Assemblies

Aptamers have emerged as versatile affinity ligands and as promising alternatives to protein antibodies. However, the inconsistency in the reported affinities and specificities of aptamers has greatly hindered the development of aptamer-based applications. Herein, we present a strategy to characterize aptamers by using DNA origami-based chiral plasmonic assemblies as reporters and establishing a competitive hybridization reaction-based thermodynamic model. We demonstrate the characterization of several DNA aptamers, including aptamers for small molecules and macromolecules, as well as aptamers with high and low affinities. The presented characterization scheme can be readily adapted to a wide selection of aptamers. We anticipate that our approach will advance the development of aptamer-based applications by enabling reliable and reproducible characterization of aptamers.


Section 1. Materials and methods
1.1. Materials DNA scaffold strands (p7650) were purchased from tilibit nanosystems; staple strands from ThermoFisher; thiol modified T16 DNA strands from Biomers; other DNA strands from IDT. Buffers and other chemicals were purchased from Fisher Scientific or Sigma-Aldrich. All reagents are commercially available and were used without any further purification. Type I ultrapure deionized (DI) water from the Milli-Q system was used for all experiments.     The origami structures were purified using centrifuge filters with the molecular weight cut off (MWCO) of 100 kDa following the instruction provided by the manufacture (Milipore). The concentration of origami structures was calculated by measuring the absorbance at 260 nm using the extinction coefficient 1.3•10 8 M -1 cm -1 . The purified origami structures were stored at 4 °C in DNA LoBind tubes after purification. To examine the origami structures, 10 μL samples were run through a 2% agarose gel in 0.5× TBE buffer with the supplement of 10 mM MgCl2. The gel was run at 100 V for 4 h. The Sybrgold was used as the staining agent and the gel was imaged with the Bio-Rad Gel Doc XR system.
Assembly of DNA origami-gold nanorods Gold nanorods (AuNRs) were synthesized following the protocol adopted from literature. 5,6 For assembly of AuNRs of DNA origami templates, thiolated DNA strands were first attached to AuNRs using the procedure described in the previous literature. 7,8 The free thiol-DNA was washed away by centrifugation at 7k rcf for 30 min for 4 times. The DNA strands on the AuNRs hybridized with the extended sequence of the staple strands to anchor the AuNRs on the origami. The AuNR-DNA and origami were mixed with 15:1 ratio and annealed from 40 °C to room temperature. To purify the samples, the origami-AuNRs were loaded into a 0.7% agarose gel with 13 mM MgCl2. After running the gel electrophoresis at 80 V for 3 h with ice cooling, the origami-AuNRs band was cut and extracted. The concentration of origami-AuNRs constructs was calculated by measuring the absorbance at maximum peak (at ~650 nm) with an estimated extinction coefficient of 3.8•10 9 M -1 cm -1 . The purified origami-AuNRs constructs (see Figure S5) were stored at 4 °C.

Microscopy and spectroscopy characterization Transmission electronic microscopy (TEM)
The origami-AuNRs constructs were imaged using FEI Tecnai F12 electron microscope operated at 120 kV. To deposit origami-AuNRs constructs on grid, 5 μL of the sample solutions was adsorbed onto a glow discharged carbon-film-coated copper grids for 8 min, followed by staining with a 1% uranyl formate solution containing 25 mM NaOH for 20 s.

Circular dichroism (CD) measurements
The origami-AuNRs, which employed a pair of aptamer and complementary strand, were incubated in 70 μL buffers (1×PBS sumplemented with MgCl2 (5mM)) with/without analyte for overnight at room temperature with shaking. The concentrations of the origami-AuNRs constructs were between 15 and 50 pM. The analyte concentrations of ATP, glucose, and thrombin were 1 mM, 100 mM, and ~170 nM (20 units mL -1 ), respectively. The control analytes (GTP/CTP/UTP, fructose, protein markers of different sizes) were used at the same or higher concentration as the target analytes. The CD spectra and extinction spectra were measured using Jasco J-1500 CD spectrometer.

Reproducibility
Affinity and specificity characterization workflow was repeated twice starting from staples, scaffold and AuNRs. The outcomes of two independent experiments were consistent.

Section 2. Analytical model and data analysis 2.1 Analytical model Concentrations at equilibrium
, where, A, B and C are aptamer, analyte, and complementary strand, respectively. Of note, although the replacement reaction (reaction 3) is present in the system, KD1 (reaction 1) and KD2 (reactions 2) are sufficient to determine the concentration of each spices at equilibrium (Table S4). In Table  S4, 0 stands for the total input local concentration of A strand, which is equal to the total input local concertation of C strand; 0 is the total input bulk concentration of analyte (B). As the amount of B (≥10 pmole) is much larger than the amount of A (<1 fmole), the concentration of analyte at equilibrium approximately equals to the initial concentration 0 . For a fixed aptamer and complementary strand with the hybridization length of n base pair, is the concentration of the AC hybrid in the absence of analyte; and are the concentrations of AC hybrid and the AB aptamer-analyte complex in the presence of the analyte, respectively. The concentrations of the other species were calculated from the mass conservation.  [AC] (S1) [AB]

Data analysis
To obtain : Gibbs free energy of the DNA hybridization in the origami-AuNRs construct (∆ r°) may differ from the Gibbs free energy calculated from the mfold software (∆ theory°) , which uses the data in the free solution. Thus, a correction coefficient was introduced, To simplify further derivation, we define: 1 = / . R is the gas constant and T is the Kelvin temperature. Therefore, We used a template strand (A*) replacing the aptamer strand for calibration. The template strandcomplementary strand hybridization length (n) was varied in a set of the origami-AuNRs constructs. Series of ∆ theory°( ) were obtained from mfold. 9 The normalized CD signals ( ) were obtained from the measurements to calculate the fraction of the origami-AuNRs constructs in the closed configuration ( ). and depends on the hybridization length between the aptamer and the complementary strand (n, see Figure 1 in the main text and Figure S2). In principle, both closed and open can be obtained from experimental measurements. However, it is technically challenging to ensure that all the origami-AuNRs constructs at the closed configuration. Hence, was obtained by fitting ∆ theory°( ) dependence on (with n={8, 9,10,11,12,13,14}) with closed being another fitting parameter. From the fitting, the coefficient was determined as 3.76±0.373 kJ·mol -1 with R 2 =0.988, closed as 671.2, and value as 0.65 which is comparable with previous works. The assumption that the Gibbs free energy of the system can be corrected by introducing was verified by the linearity between ∆ theory°( ) and the ( ) (see Figure S3). S18 Figure S3. The linear fitting of ∆ theory°( ) dependence on ( )

To obtain :
The ratio of the relative normalized CD signal in the presence and absence of the analytes, equals to the ratio of AC concentrations in the presence and absence of the analyte ( ) and, according to equation S26, can be obtained from CD and Abs measurements for the following samples: 1) The origami-AuNRs constructs with aptamer and complementary strand in the absence and presence of analyte ( 0 =0 , 0 ); Usually n is varied with 4-5 values, giving 8-10 samples.
2 To obtain 0 , we prepared 4 constructs with n varied from 10 to 13 bp. The corresponding D1 ( ) were calculated using ∆ ℎ° obtained from the software mfold according to equation S37. We measured the CD and Abs signals for each construct in the presence and absence of a DNA competitor strand with the known D2 (2.28 μM) to gain . The input local concentration ( 0 ) was obtained as 76±3.8 μM, by fitting with D1 ( ) using equation S38, in the fixed concentration 0 (12.5 μM), R 2 =0.995 ( Figure 2E in the main text).     To obtain specificity: The fixed aptamer and complementary strand pair were chosen based on the affinity experiment so that the kinetic trap and side product were avoided. The origami-AuNRs with the fixed aptamer and complementary strand was incubated with different analytes or without any analyte and the normalized CD signal ( ) was obtained. The normalized signal of the origami-AuNRs in buffer was set as 100% and the normalized signal of the origami-AuNRs with different analyte treatment was divided by the normalized signal in buffer to gain the percentage. The signal drop percentages in different analytes were compared to qualitatively determine the specificity.
S22 Figure S4. The workflow of the aptamer characterization.

Section 3. Design considerations and benefits of plasmonic chiral probes for the aptamer characterization
Directly measuring the fractions of bound and unbound aptamers relies on finding specific characteristics to differentiate the analyte-aptamer complex and free aptamers, which often change from case to case, and thus dictates different approaches. However, by applying competitive hybridization reactions-based strategy, a unified technique can be applied to various aptamers.
Compared to the traditional model in competitive hybridization reactions-based strategy, where concentration dependency is used with a fixed complementary strand, our approach varies the complementary strands both in hybridization region and lengths at a fixed analyte concentration and is expected to be more generalizable.
Considering the heterogeneous nature of the aptamer domains, typically the whole aptamer sequence can be sepearated into three functional domains (non-essential, essential but non-critical, and critical domains): i) The non-essential domain contains the sequence that neither interacts with the analyte nor supports structural folding and should be truncated; ii) The essential but non-critical domain contains the sequence which is not critical for the initial interaction of analyte but essential for analyte binding; iii) The critical domain contains the sequence that plays important role in the initial interaction of analyte. Choosing a complementary strand without preliminary knowledge of the aptamer domains suffers from the risk of forming kinetic traps and/or side product, causing unreliable affinity ( D ) measurement. Specifically, a complementary strand hybridizing with the non-essential domain of the aptamer, can form the stable analyte-aptamer-complementary strand complex and, consequently, prevent the separation of the aptamer and the complementary strand. In addition, a complementary strand, which blocks the critical domain, can kinetically trap the aptamer at the hybridized state by jeopardizing the induced-fit path, and, consequently, hinders the analyte binding reaction reaching equilibrium when the dissociation rate is slow. By varying the complementary strands and using the goodness of fitting (R 2 ), the reliability of the D measurements can be ensured. Only when the equilibrium is reached and no side product is generated, the ( , D1 ( )) can fit the model well.
To set a meaningful threshold of R 2 for the judgment of the validity, adjusting values into a proper range is required. If most are close to 1 (CD signals remaining similar to the original levels after the addition of the analyte), the fitting might generate an invalid D2 value with a high R 2 just because the mathmetical calculation of the deviation is small. If most of values are close to 0 (CD signals droping to 0 after the addition of the analyte), the fitting might generate a valid D2 value with a low R 2 because the system erros become large when the signals are small. Therefore, as = ( 0 , 0 , D1 , D2 ), we typically used the fixed concentration of analyte ( 0 ) at approximately 10-100 times of the D2 value so the values of fall into the range of 0.2-0.99. The hybridization length ( ) of the aptamer and complementary strand falls into the 7-12 bp range to ensure the initial signal is large enough while the two state model of DNA hybridization and separation is still valid. The complementary strands used in this work hybridize either to 5' or 3' end of aptamers as the valid D2 values have already been obtained from one end. However, in principle, the complementary strands can map over the whole aptamer when the essential but non-critical domain is not contained by the end sequence. The appraoch is expected to be generalizable because in principle, all aptamers should contain an essential but non-critical sequence. The 'aptamer' that only contains non-essential sequence is not an aptamer. There is, however, a possibility that the aptamer only contains the critical domain so introducing any complementary strands will block the induced-fit path and create a kinetic trap. To solve the issue, after obtaining the information of the aptamer domain, a fixed short complementary sequence (8-9 bp), which allows fast dissociation, together with the concentration dependency fitting can be used as we have demonstrated in the Figure 3D. In some specific cases, where the aptamers have significant amount of base pairs (e.g. RNA aptamer against ATP, RNA aptamer against HIV-1 Tat, DNA aptamer against cocaine etc.), the hetero-hybridization has to compete against the self-hybridization to form the hybrid. To keep the Gibbs free energy of the hybridization reaction similar to the general cases, a more stable hybridization between the aptamer and the complementary strand is required for these special cases as the Gibbs free energy of the product has to be low to compensate for the low Gibbs free energy of the reactants. Consequently, the slow dissociation rate problem arises here. In this case, the complementary strand allowing the induced-fit path has to be guaranteed. In rare cases, the complementary strand may act as a partial split aptamer as it contains the same or similar sequence of the parent aptamer and thus fail to be competed off by the analyte binding. In this scenario, the competitive hybridization reaction-based model should be replaced by the sandwich model of the split aptamer and analyte. The plasmonic chiral probes provide the feasibility of the approach of varying complementary strands to the aptamer due to the high signal-to-noise ratio, allowing the system to differentiate the change of a single base pair ( Figure 2B). The traditional fluorescence-based probes rely on a stable binding between aptamer and its complementary strand to reduce the background noise, which may hinder the system from reaching equilibrium. The common approach to solving this kinetical trap problem is the addition of extra nucleotides to the aptamer sequence to provide a longer hybridization length for the complementary strand while maintaining the critical domain accessible by the analyte. This, however, may cause the side product of the analyte-aptamer-complementary strand complex and is only possible for the hybridization starting from the ends of the aptamer.