Integration of an Expression Platform in the SELEX Cycle to Select DNA Aptamer Binding to a Disease Biomarker.

Aptamers can be developed for biosensors, diagnostic tools, and therapeutic reagents. These applications usually require a fusion of aptamers and expression platforms. However, the fusion process is usually time-consuming and laborious. In this study, we integrated the deoxyribozyme (I-R3) as an expression platform in the SELEX cycle (called Expression-SELEX) to select aptazymes that can sense diverse molecules. We used the Maple syrup urine disease (MSUD) biomarker L-allo-isoleucine to test the selection model. After five rounds of screening, the cleavage products were sufficiently enriched to be visualized on polyacrylamide gel electrophoresis (PAGE) gel. Through high-throughput sequencing analysis, several candidates were identified. One such candidate, IR3-I-DNA, binds L-allo-isoleucine with a dissociation constant (K D) of 0.57 mM. When the ligand was present, the cleavage fraction of IR3-I-DNA increased from 0.3 to 0.5, and its K obs value improved from 1.38 min-1 to 1.97 min-1. Our selection approach can also be applied to produce aptazymes that can bind to variable ligands and be used more directly as biosensors.

Introduction

Aptamers are single DNA or RNA molecules that fold in specific secondary and tertiary structures to bind to target molecules (ligands) with high specificity and affinity.[1,2] Aptamers were first invented by researchers in 1990 while selecting RNAs that can bind to T4 DNA polymerase, organic dye molecules, or other ligands.[1−3] This procedure is called the systematic evolution of ligands by exponential enrichment (SELEX), in which a DNA library with a large population by including random sequences is built and a filter containing immobilized ligands is used to screen for DNA or RNA molecules (aptamers) that can specifically recognize and attach to ligands. These aptamers have been amplified by PCR or RT-PCR and subjected to another selection next cycle. Commonly, after 12–14 cycles, the aptamers that bind to ligands with high specificity and affinity can be obtained.

Since the invention of SELEX, many aptamers have been discovered and applied in various research fields, including biosensors,[4−7] gene-expression regulators,[8−11] and therapeutic reagents.[12] Two well-known aptamers that are applied as biosensors and gene-expression regulators are the theophylline aptamer and the VEGF aptamer.[12,13] Theophylline is a natural drug used to treat asthma, bronchitis, and emphysema; however, serum levels must be monitored carefully to avoid serious toxicity.[13] Theophylline is also chemically similar to theobromine and caffeine, which are also present in serum samples.[13] A theophylline aptamer that binds specifically to theophylline with high affinity and discriminates efficiently against its analogues has been developed to monitor theophylline in the serum.[13] Additionally, the theophylline aptamer has been applied for gene regulation in living cells.[11,14] Another well-known aptamer is the Pegaptanib aptamer, which binds vascular endothelial growth factor (VEGF) and has been approved by the Food and Drug Administration (FDA) to treat macular degeneration in 2004. Therefore, Pegaptanib became the first aptamer therapeutic approved for use in humans, paving the way for future aptamer applications.

Many SELEX-related technologies, such as negative SELEX,[15] counter SELEX,[13] capillary electrophoresis SELEX (CE-SELEX),[16] Cell SELEX,[17] in vivo SELEX,[18] and Capture-SELEX,[19] have been developed during the discovery of new aptamers. The major purposes of these technologies are to eliminate background,[15] increase the ligand-binding specificity,[13] improve the screening efficiency,[16] target membrane proteins on the cell,[17] bind ligands in the mouse tumor in vivo,[18] and detect structural switching signals during ligand binding.[19]

Many aptamers produced by SELEX or SELEX-related technologies have been attached to various platforms to conduct specific functions upon ligand binding.[5,20−22] Breaker and co-workers have attached ATP, flavin mononucleotide (FMN), and theophylline aptamers to hammerhead ribozymes (HHR)[5,23] to make allosteric ribozymes. The existence of natural FMN aptamers[24] and other metabolite-binding aptamers has then been proven in bacteria that can cooperate with expression platforms to regulate gene expression.[25−28] These natural regulatory aptamers are termed riboswitches. One of the riboswitches uses ribozyme as an expression platform (the glmS ribozyme),[29] while other riboswitches use ribosome binding sites, terminators, poly(A) processing sites, or splicing regions as expression platforms.[27,30−32] Since then, a plethora of aptamers has been fused to expression platforms to regulate gene expression. For example, Hartig and other groups have attached aptamers to ribozymes and other platforms and transformed them into bacteria,[8,33] yeast,[9,22,34] and mammalian cells[21] to successfully regulate gene expression by affecting transcription elongation, translation initiation, or mRNA stability.

However, the fusion between the aptamer and the ribozyme (or other expression platforms) is time-consuming and labor-intensive. We intend to integrate the fusion process with SELEX cycles. We call this process Expression-SELEX, which includes random sequences in the aptamer region and can select aptamers that can bind to ligands of interest. As the aptamers carry an expression platform, they can be developed into biosensors if the allosteric aptazymes are labeled with a fluorescent group and they can be gene expression regulators if they are placed in the 3′ UTR or 5′ UTR of gene constructs.

In this study, we have selected DNA allosteric aptazymes that bind to a ligand and induce self-cleavage. The integrated deoxyribozyme is the highly active self-cleaving deoxyribozyme I-R3,[35] which has been recently produced by Breaker’s lab. We used l-allo-isoleucine, which is a biomarker of maple syrup urine disease (MSUD), as a ligand.[36] However, our selection approach can also be applied to select aptamers that bind to different ligands, such as biomarkers of other diseases.

Results

Selecting Novel Aptamers That Fuse with a Deoxyribozyme

Since the invention of SELEX, many aptamers have been generated by screening for single-stranded DNAs or RNAs (aptamers) that bind to different molecules (ligands). These aptamers have been widely used for constructing biosensors, diagnostic tools, and therapeutic reagents after the integration of different expression platforms that can conduct different activities. However, the fusion process is time-consuming and labor-intensive. Here, we attempt to integrate an expression platform (such as a deoxyribozyme) in the SELEX process to generate novel aptamers that can bind to different ligands and conduct activities, such as self-cleaving after binding to cognate molecules (Figure ). As the aptamer region and the deoxyribozyme region overlap, the binding signals can transfer to the deoxyribozyme region and result in DNA cleavage. The cleavage will release these aptazymes from the streptavidin-beads that use docking oligonucleotides to base-pair with the linker region of the DNA library (Figure ). The released ssDNA is recovered for the next cycle of selection and enrichment.

Figure 1

Fusion of a deoxyribozyme with a DNA aptamer for Expression-SELEX. The stem P2 of the deoxyribozyme I-R3[35] is replaced with a random N60 sequence. The red bases are conserved nucleotides of I-R3, the orange line denotes the docking oligonucleotides with biotin, and the red line is the linker sequence that base pairs to the docking sequence to immobilize the DNA.

The selection steps are described in the legend of Figure . Briefly, the DNA library, containing full-length DNAs that would not split without a ligand, is incubated with 100 μM l-all-isoleucine in cleavage buffer for 20 min. The 5′ cleavage product is collected and restored to the full-length double-strand DNAs by PCR. The double-strand DNAs are denatured and segregated to obtain single-strand DNA for the next screening cycle. The important feature of this SELEX is the combination of the aptamer (random sequences) with the expression platform to facilitate the selection process and the application of the aptamer.

Figure 2

Expression-SELEX for selecting novel DNA aptamers integrated with a deoxyribozyme. Step 1: The single-stranded DNA library carrying linker sequences was immobilized by base-pairing to docking oligonucleotides on the beads and rinsed to remove the DNA that cleaves in the absence of a ligand. Step 2: The full-length DNAs on the beads were incubated with the ligand and Zn2+. The DNA that could bind to the ligand and self-cleave was released into the buffer. Step 3: The 5′ cleavage product was purified from the supernatant by polyacrylamide gel electrophoresis (PAGE) gel purification. Step 4: The 5′ cleavage product was amplified by PCR using a pair of primers that could restore the full-length DNA. Step 5: Single-stranded DNA was prepared either by NaOH denaturation or by asymmetric PCR. These steps were repeated until an aptamer that could bind tightly to the ligand was selected.

We use a library consisting of 1015 ssDNA, with a length of 113 nucleotides (nt), including a random sequence of 60 nt in the middle (Figure ) for Expression-SELEX. After these ssDNA molecules are immobilized to the beads, we incubate them with the cleavage buffer without the ligand and removed the ssDNA molecules that undergo self-cleavage. The remaining full-length ssDNA molecules are incubated with the ligand in the cleavage buffer. The 5′ cleavage products of the ssDNA molecules are harvested from the supernatant and run on PAGE gel for purification (Figure a). Two markers are loaded to trace the precursor and the cleavage product. The area corresponding to the 86 nt is cut out and purified. The purified cleavage products are restored to the full-length ssDNA for the next cycle. After five cycles, the enriched 5′ cleavage products of ssDNA molecules appear to be a clear band on the PAGE gel (Figure b).

Figure 3

Enriched DNA cleavage products. (a) In the first round of screening, the 5′ cleavage product on the polyacrylamide gel electrophoresis (PAGE) gel was barely visualized. (b) In the fifth round of screening, the 5′ cleavage product was enriched and visible on the PAGE gel. Pre: Full-length single-strand DNA. Clv: 5′ cleavage product.

To investigate the sequences of the allosteric deoxyribozyme candidates enriched in the Expression-SELEX cycles, the 5′ cleavage products of the fifth round are attached to the P5 and P7 adaptors and amplified by PCR to build a library for Illumina sequencing. The results show many sequences in the library, some of which are enriched. We analyze the secondary structures of these ssDNA sequences by Mfold.[37] Then, we test the cleavage activity of these aptamer candidates and find that IR3-I-DNA (Figure a) has a good cleavage activity. The aptamer part of the IR3-I-DNA forms an “L” shape with three internal loops.

Figure 4

Binding specificity of the IR3-I-DNA aptamer candidate to l-allo-isoleucine. (a) The sequence and the secondary structure of IR3-I-DNA. The aptamer part is an “L” shaped DNA with three internal loops. The IR3-I-DNA aptamer was incubated in the cleavage buffer with different ligands for 1.5 min at 37 °C. The docking oligonucleotides were also included in the reaction to the base pair with the linker sequence. (b) The cleavage products were separated by polyacrylamide gel electrophoresis (PAGE) gel and stained with SYBR Gold (Invitrogen). The fraction of DNA cleavage was the 5′ and 3′ cleavage products (band intensities) divided by the sum of the remaining full-length DNA, and 5′ and 3′ cleavage products (band intensities). The precursor (Pre), the 5′ cleavage (5′ Clv) and 3′ cleavage (3′ Clv) products, and docking oligonucleotides are shown in the figure. The experiment was repeated twice with similar results, and a representative image is shown. (c) Structures of the ligands l-allo-isoleucine, isoleucine, and leucine. Allo-iso-leu, iso-leu, and leu represent the ligands l-allo-isoleucine, isoleucine, and leucine with a concentration of 2.5 mM.

Next, the IR3-I-DNA (Figure a) is incubated in the cleavage buffer with l-allo-isoleucine, isoleucine, and leucine for 90 s at 37 °C. Without a ligand, the fraction of DNA cleavage is 0.35 and increases to 0.51 when allo-isoleucine is present (Figure b). The analogues of l-allo-isoleucine, such as isoleucine and leucine (Figure b,c), also induce cleavage. The different inductions of cleavage by these analogues can be observed, although they are small differences, likely due to the close similarity between these molecules.

To test the induction effect of the ligand on the cleavage of the allosteric DNA ribozyme, we conduct the time-course experiment of the DNA cleavage by incubating IR3-I-DNA with a cleavage buffer with and without a ligand at different time points. The result show that without a ligand, the slope of the cleavage curve is not as steep as the one with the ligand (Figure ). The cleavage fraction is about 0.35 (ratio of the cleavage products divided by the total RNA) without the ligand, and it is 0.5 (increasing 43%) when the ligand is present (Figure c). The Kobs values have been estimated by GraphPad Prism. Without a ligand, the Kobs value is 1.38 min–1, which increases to 1.97 min–1 in the presence of ligand (1 mM). This result suggests that the cleavage speed of the IR3-I-DNA allosteric ribozyme is fast and that the ligand can induce the cleavage of IR3-I-DNA.

Figure 5

Induction of cleavage by l-allo-isoleucine on the IR3-I-DNA allosteric deoxyribozyme. IR3-I-DNA was incubated in the cleavage buffer with and without a ligand for different time points. The fraction of cleavage was calculated as described in Figure . M1 and M2 are ssDNA markers of 113 nt and 86 nt, respectively (Table S1). (a) Without a ligand. (b) With the ligand l-allo-isoleucine. (c) The values of Kobs were measured by the time-course experiments from 10 s to 2 h. The experiment was repeated three times with similar results and a representative is shown.

To further investigate the induction of the cleavage by the ligand, we incubate IR3-I-DNA with different concentrations of l-allo-isoleucine for 90 s. The PAGE gel analysis shows that with the increase in ligand (Figure a), the fraction of DNA cleavage increases gradually from approximately 30% to 50% (Figure b), increasing 66% when the ligand concentration is increased from 10 μM to 2.5 mM. The KD value of the IR3-I-DNA is 570 μM. This result suggests that the cleavage can be induced by the ligand at concentrations >10 μM.

Figure 6

Binding affinity of l-allo-isoleucine by the IR3-I-DNA allosteric deoxyribozyme. IR3-I-DNA was incubated in cleavage buffer with different concentrations of the ligand for 90 s. The fraction of cleavage was calculated as described in Figure a. (a) Polyacrylamide gel electrophoresis (PAGE) analysis of the cleavage of IR3-I-DNA allosteric deoxyribozyme with concentrations of allo-l-isoleucine ranging from 100 to 2.5 mM. Labels are as described in Figure b. (b) Dissociation constants (KD) of IR3-I-DNA. The KD values were measured from three repeats of PAGE gel assays, with the logarithm of the concentration (c) of ligand in molar units.

Discussion

Since the invention of the SELEX technology, many aptamers have been produced and used widely as biosensors, diagnostic tools, and therapeutic reagents. Among these aptamers, the VEGF aptamer (Pegaptanib) has been approved by the FDA to treat macular degeneration.[12]

Fusion of an Aptamer to an Expression Platform

The original SELEX technology commonly takes a long time to obtain an aptamer, and the success rate is low.[38] Many new SELEX approaches have been invented, such as CE-SELEX[16] and Capture-SELEX.[19] These approaches use random sequences to catch target molecules, ranging from single nucleotides, such as ATP to proteins. Before these aptamers can be used as biosensors or gene regulators, they need to be fused with an expression platform,[34,39] for example, the attachment of the TPP, FMN, theophylline, and neomycin aptamers to HHR to regulate gene expression.[9,23,40] However, the fusion process is usually time-consuming and labor-intensive. We have attempted to establish the Expression-SELEX to select allosteric deoxyribozymes that bind to various molecules and induce cleavage of the deoxyribozyme.

Deoxyribozyme Not Only Acts as an Expression Platform but Also Facilitates the Selection Process

An important step in the SELEX is to select the aptamers that bind to the ligand. The original method involves different buffers to elute the aptamers from the ligand immobilized on the column.[1,2] Capture-SELEX uses docking oligonucleotides to immobilize candidate aptamers and release candidates only when ligand binding changes the structure of the aptamer.[19] In this study, if a ligand binds to the aptamer candidates and induces the cleavage of the deoxyribozyme, the aptamer would be liberated from the beads. Thus, the deoxyribozyme we integrated into the SELEX cycle has a dual function.

IR3-I-DNA Is a Ligand Inducible, Fast Cleaving Dexoyribozyme

Through five cycles of the Expression-SELEX, a clear DNA band appears on the PAGE gel with the expected size. The high-throughput analysis by Illumina sequencing and DNA Mfold[37] shows that some of the aptamer candidates can form stem-loop structures in the aptamer region and a bridge between the aptamer region and the deoxyribozyme region. We have performed a cleavage assay for some of them and have found that IR3-I-DNA can self-cleave and respond to the ligand l-allo-isoleucine.

We have performed an enzymatic assay using 100–400 ng (approximately 3–13 pmol) of IR3-I-DNA deoxyribozyme and 4 mM Zn2+ to initiate the cleavage assay (Materials and Methods). I-R3 deoxyribozyme can also be separated into a substrate part and an enzyme part to measure enzymatic activity.[35] However, for the IR3-I-DNA allosteric deoxyribozyme, it is convenient to analyze the full activity of the entire enzyme (fusion of the aptamer and ribozyme); additionally, it is not necessary to determine where to cut this DNAzyme into two parts.

The specificity test shows that the induction of cleavage by isoleucine is very close to that for l-allo-isoleucine, which may be due to the close similarity between these two molecules. The induction by leucine is a little less than that for l-allo-isoleucine, indicating that the DNA aptamer can distinguish between l-allo-isoleucine and leucine; these compounds differ only in their conformations.

Regarding the kinetic mode of the induction of cleavage by l-allo-isoleucine on the IR3-I-DNA allosteric deoxyribozyme, the ligand (l-allo-isoleucine) binds to the aptamer of the allosteric deoxyribozyme and causes the conformation of the aptamer to change to form a stable stem between the aptamer and the I-R3 deoxyribozyme. The stable stem facilitates the folding and self-cleavage of the IR3-I-DNA allosteric deoxyribozyme. The percentage of allosteric DNAzyme self-cleavage increases with increasing incubation time (Figure c).

The binding affinity for IR3-I-DNA when binding to l-allo-isoleucine has been measured by KD with a value of 570 μM, while the cleavage induction by l-allo-isoleucine binding can be up to 66% (Figure b) when the ligand concentration increases from 10 μM to 2.5 mM. These results suggest that the ligand can bind to the aptamer and cleavage can be induced by the ligand. The cleavage speed is also affected by the ligand binding. Indeed, the Kobs value of IR3-I-DNA increases from 1.38 to 1.97 min–1 (an increase of 66%) when the ligand is present, suggesting that the cleavage speed of the IR3-I-DNA allosteric ribozyme is fast and that the ligand can induce the cleavage of IR3-I-DNA. However, we also observe some background cleavage signals even in the absence of ligand. This defect can be reduced in the selection cycles by increasing the incubation time during the negative selection reaction and decreasing the incubation time for the positive selection reaction to disfavor slow-cleaving ribozymes.[41] Additionally, to obtain an aptamer candidate with better specificity and higher affinity, one can use counter-selection, more cycles of screening, a lower concentration of ligand, and a shorter incubation time.

Influence of Metal Ions on the Cleavage of IR3-I-DNA Deoxyribozyme

Among divalent cations, such as Cd2+, Co2+, Cu2+, Ni2+, Mn2+, Ca2+, and Mg2+, only Zn2+ supports I-R3 deoxyribozyme activity.[35] We have not tested the influence of metal ions on the enzyme activity of the IR3-I-DNA allosteric deoxyribozyme. However, we expect that this deoxyribozyme also requires Zn2+ for its ribozyme activity and that other cations cannot support its enzyme activity given that IR3-I-DNA contains the majority of the I-R3 deoxyribozyme.

Aptazymes Such as IR3-I-DNA Can Be Attached by a Fluorescent Group to Become Biosensors

The 5′ cleavage product of the IR3-I-DNA can be easily detected by SYBR Gold staining within the PAGE gel to sense the ligand concentrations. IR3-I-DNA is functionally a biosensor. Alternatively, a cyanine fluorescent dye, such as cy3, cy5, or cy7, can be attached to the 5′ end of IR3-I-DNA and the biotin to its 3′ end and then immobilize the DNA to streptavidin beads. Consequently, the cleavage of IR3-I-DNA induced by the ligand binding can be detected from the 5′ cleavage products in the supernatant by the cyanine fluorescence intensity.

Conclusions

The selection of aptamers by SELEX and the engineering of aptamers as a biosensor or gene regulator by fusing an expression platform to the aptamers are commonly two separate processes. In this study, we integrate a deoxyribozyme as an expression platform in the SELEX cycle to select aptazymes that can bind to a ligand and self-cleave upon ligand binding. Thus, we merge these processes in one cycle, termed Expression-SELEX. After five cycles of screening, we identify an aptazyme, IR3-I-DNA, that can bind to l-allo-isoleucine with a dissociation constant (KD) of 0.57 mM. The induction of the aptazyme cleavage is up to 66% when the ligand is present and has an observed rate constant (Kobs) of up to 1.97/min. These results suggest that the Expression-SELEX would enable the production of aptazymes with integrated expression platforms to be more directly used as biosensors or gene regulators.

Materials and Methods

Expression-SELEX Procedures

The single-stranded DNA (ssDNA) library I-R3-random (Table S1) containing about 1015 molecules was purchased from Genewiz (Suzhou, China). Step 1: The first round was initiated with 2 nmol of ssDNA (2 × 1014 molecules), with the sequence and structure shown in Figure . These molecules were incubated with the same number of docking-oligonucleotides (modified with biotin) (Table S1) in HEPES buffer I (0.1 M HEPES, 0.1 M NaCl, pH 7.05 at 23 °C) under a heat-shock program (80 °C for 2 min, 60 °C for 2 min, 45 °C for 2 min, and 37 °C for 2 min) to anneal the docking-oligonucleotides to the ssDNA. Then, the DNA library was immobilized to the beads with streptavidin (PuriMag, China) following the manufacturer’s protocol (50 μL of 10 mg/mL beads can bind to 125 pmol of ssDNA). The immobilized ssDNA was rinsed with HEPES buffer I three times and dissolved in 50 μL of 2× HEPES buffer I. Then, 50 μL of 2× HEPES buffer II (0.2 M HEPES, 0.2 M NaCl, 8 mM MgCl2, and 2 mM ZnCl2, pH 7.05 at 23 °C) was added to the above ssDNA mixture, and the mixture was incubated at 37 °C for 4 h. Next, the beads were rinsed with the same buffer twice more to eliminate self-cleaved ssDNA. Step 2: The beads were incubated with 200 μL of buffer II containing 0.1 mM l-all-isoleucine at 37 °C for 20 min. The supernatant was collected, and the cleaved ssDNA was precipitated with 75% ethanol and 0.3 M NaAc. Step 3: The 5′ cleavage product of the ssDNA was purified by 10% denaturing PAGE (containing 8 M Urea), isolated from the gel by crush-soaking in a solution containing 10 mM Tris–HCl (pH 7.5 at 23 °C), 200 mM NaCl, and 1 mM EDTA, precipitated by the addition of 2.5 volumes of 100% cold ethanol, recovered by centrifugation, and resuspended in ddH2O. Step 4: The purified cleavage product was amplified by PCR using the primers IR3-fwd and IR3-rev1 (Table S1). The PCR product was purified by agarose gel purification using SanPrep (Shengong, China). Step 5: The purified PCR product was used to produce ssDNA using the method of asymmetric PCR.[42] Specifically, 100 ng of above purified PCR was used as a template, and 0.24 μM IR3-fwd and 0.004 μM of IR3-rev1 (Table S1) were used as primers for 30 PCR cycles. The ssDNA was isolated and purified by PAGE gel as described above. The product of this ssDNA was used for the next cycle of Expression-SELEX.

Next-Generation Sequencing (NGS) of the Enriched Aptamers from SELEX

The PCR product from the fifth round of selection was purified by a column purification kit (Sangon Biotech). For NGS library preparation, the P5 and P7 adaptors (Table S1) from the Illumina platform were added to the end of the DNA fragments by PCR. Specifically, 100 ng of purified DNA, 0.2 μM P5-forward primer, 0.2 μM P7-Reverse primer, and 2× PCR Taq MasterMix were mixed in a 100 μL volume. The PCR cycles included a denaturation cycle at 95 °C for 3 min, 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s and a final extension at 72 °C for 3 min. The PCR product was purified and sent to Beijing Novegene Company for high-throughput sequencing.

DNA Aptazyme Cleavage Assays

Cleavage assays were performed under conditions similar to those described previously.[43] Briefly, 100–400 ng of DNA was incubated with or without the ligand l-allo-isoleucine in cleavage buffer (0.2 M HEPES, 0.2 M NaCl, 8 mM MgCl2, and 4 mM ZnCl2, pH 7.05 at 23 °C) in a final volume of 20 μL. After incubation at 37 °C for the time indicated, samples were loaded onto the 20% PAGE gel to separate the cleavage products. The ssDNA was visualized by staining with SYBR Gold (Invitrogen). The cleavage efficiency was calculated from the fraction of cleavage, which was the intensity of the 5′ and 3′ cleavage DNA products divided by the total ssDNA (cleaved and uncleaved products).

Dissociation Constant (KD) Measurements

Apparent KD values were determined by the method described previously.[6] The concentration of the ligand that can cause a 50% shift of the cleavage of IR3-I-DNA was defined as the apparent KD. The KD was calculated using GraphPad Software with the function of specific binding with Hill slope and the equation Y = Bmax × X/(KD + X), where Bmax was the maximum-specific binding in the same units as Y.

Observed Rate Constant (Kobs) Measurements

Measurements of Kobs values were performed as described previously.[43] Briefly, we performed cleavage of the IR3-I-DNA with and without a ligand in the cleavage buffer with an incubation time from 10 s to 2 h. The cleavage products were separated by PAGE gel, and the intensity of each band was quantified using ImageJ. The fraction of cleavage at each time point was calculated as described above. The Kobs for each reaction was measured using GraphPad Software with one phase decay and the equation Y = (Y0 – plateau) exp(−KobsX) + plateau, where Y0 was the Y value when X (time) was zero, plateau was the Y value at infinite times, and Kobs was the rate constant, expressed in reciprocal of the X-axis time units (minutes).