Evolution of a bispecific G-quadruplex-forming circular aptamer to block IL-6/sIL-6R interaction for inflammation inhibition

IL-6 (interleukin-6) is an essential cytokine that participates in many inflammatory and immune responses, and disrupting the interaction between IL-6 and its receptor sIL-6R (soluble form of IL-6 receptor) represents a promising treatment strategy for inflammation and related diseases. Herein we report the first-ever effort of evolving a bispecific circular aptamer, named CIL-6A6-1, that is capable of binding both IL-6 and sIL-6R with nanomolar affinities and is stable in serum for more than 48 hours. CIL-6A6-1 can effectively block the IL-6/sIL-6R interaction and significantly inhibit cell inflammation. Most importantly, this bispecific aptamer is much more effective than aptamers that bind IL-6 and sIL-6R alone as well as tocilizumab, a commercially available humanized monoclonal antibody against sIL-6R, highlighting the advantage of selecting bispecific circular aptamers as molecular tools for anti-inflammation therapy. Interestingly, CIL-6A6-1 is predicted to adopt a unique structural fold with two G-quadruplex motifs capped by a long single-stranded region, which differs from all known DNA aptamers. This unique structural fold may also contribute to its excellent functionality and high stability in biological complex media. We anticipate that our study will represent a significant step forward towards demonstrating the practical utility of bispecific DNA aptamers for therapeutic applications.


Library preparation
The DNA library contains a 19-nucleotide human sIL-6R binding aptamer flanked with two 20-nucleotide random domains and two constant domains at the 5-end and 3-end (note that the primer domain contains a recognition site for the restriction enzyme EcoRV), as shown in Figure S1b.The circular DNA library was prepared through template-assisted ligation with T4 DNA ligase.Briefly, linear DNA oligonucleotide was firstly phosphorylated as follows: a reaction mixture (60 μL) was made to contain 1.5 nmol linear oligonucleotide, 20 U PNK (U: unit), 1× PNK buffer (50 mM Tris-HCl, pH 7.6 at 25℃, 10 mM MgCl2, 5 mM DTT, 0.1 mM spermidine) and 1 mM ATP.The mixture was incubated at 37℃ for 40 min, followed by heating at 90℃ for 5 min.The circularization reaction was conducted in a volume of 400 μl, produced by adding 306 μl of H2O and 2 μl of a DNA template (LT1 or LT2, 100 μM) to the phosphorylation reaction mixture above.After heating at 90℃ for 3 min and cooling down at room temperature (RT) for 10 min, 40 μL of 10× T4 DNA ligase buffer (400 mM Tris-HCl, 100 mM MgCl2, 100 mM DTT, 5 mM ATP, pH 7.8 at 25℃) and 2 μL of T4 DNA ligase (5 U/ μL) were added.This mixture was incubated in RT for 2 h follow by heating at 90℃ for 5 min.Lastly, the ligated circular DNA pool was concentrated by standard ethanol precipitation and purified by 10% dPAGE.The concentration of the circular DNA template was determined spectroscopically.The circular aptamer CIL-6A6-1 was further characterized by HPLC and mass spec analysis (Figure S9).The HPLC spectrum shows the presence of a single peak (7.305 min), indicating high purity of the circular products.The observed mass in the mass spectrum (23223.0) of circular products matches the theoretical mass of CIL-6A6-1.

Molecular crowding SELEX
Six SELEX rounds were carried out.For the first round, the circular DNA library (100 pmol), which was denatured at 95℃ for 5 min and then cooled on ice for 15 min, was incubated with CAMBs (1×10 8 beads / mL) with rotation for 1 h at room temperature in 100 μL of 1× binding buffer (1×PBSMT, pH 7.2) containing 137 mM NaCl, 2.68 mM KCl, 8.1mM Na2HPO4, 1.76 mM KH2PO4, 1 mM MgCl2, and 0.025% Tween-20.The tube was then placed on a magnetic test tube holder, the supernatant was collected into a new tube.The collected circular oligonucleotides were incubated with CAMBs-bound human IL-6 (1×10 8 beads / mL) with rotation for 1 h at room temperature in 100 μL of 1× binding buffer.The tube was then placed on a magnetic test tube holder and the supernatant was removed.After that, the beads were washed three times Electronic Supplementary Material (ESI) for Chemical Science.This journal is © The Royal Society of Chemistry 2024 with 1× binding buffer (100 μL).Under the action of the magnet, protein-bound DNA molecules were eluted with 50 μL of ultrapure water at 95℃ for 15 min.After recovery by ethanol precipitation, the DNA was amplified by two rounds of rolling circle amplification (RCA), restricted digestion with EcoRV and circulation by DNA ligation.The RCA was typically performed in 50 μL of 1× RCA buffer (made from 10× stock, which is made of 330 mM Tris-acetate, pH 7.9 at 37℃, 100 mM magnesium acetate, 660 mM potassium acetate, 1% (v/v) Tween-20, 10 mM DTT) containing the DNA derived from last step, 2 μL LT1 (100 pmol), 1 mM dNTP.After heating at 90℃ for 3 min, the solution was cooled at room temperature for 10 min.Then, 0.5 μL of phi29 DNA polymerase (10 U/μL) was added, followed by incubation at 30℃ for 30 min before heated at 65℃ for 10 min to deactivate the polymerase.Subsequently, 2 μL of 500 μM LT2 was introduced to the RCA reaction mixture above.The mixture was heated at 90℃ for 3 min and cooled at RT for 10 min.Finally, 10 μL of 10× Fast Digestion Buffer (100 mM Tris-HCl, pH 8.0, 50 mM MgCl2, 1 M NaCl, 1 mg/ml BSA) and 5 μL of Fast Digest EcoRV (unit size 400 reactions; the total volume is 400 μL) were added into above mixture and the final reaction volume was increased to 100 μL.The reaction mixture was then incubated at 37℃ for 8 h, and heated at 65℃ for 10 min to inactivate the restriction enzyme.The amplified monomerized RCA products were purified by 10% dPAGE.The DNA was then eluted from the gel and circularized into circular DNA template B (CDTB) for the second RCA reaction.The reaction condition was identical to the first RCA except for the replacement of LT1 with LT2.For the restriction digestion after RCA, LT2 was replaced with LT1.To create a molecular crowding environment, human serum was introduced into the binding buffer for rounds 2-6, and the amount of serum was progressively increased from 5% (round 2) to 10% (round 3), 20% (round 4), 50% (round 5) and 50% (round 6).The round-6 DNA pool was used for deep sequencing.

Sequencing protocol
The eluted DNA in round 6 was digested into linear DNA sequences as previously described.2 μL of 0.05 μM digested DNA was amplified by two PCR steps.In PCR1, a 50 μL reaction mixture was prepared to contain the above DNA, 0.4 μM forward primer (F1) and 0.4 μM reverse primer (R1, their sequences are provided in Supplementary Table S1), 200 μM dNTP (dATP, dCTP, dGTP and dTTP), 1 × PCR buffer (75 mM Tris-HCl, pH 9.0, 2 mM MgCl2, 50 mM KCl, 20 mM (NH4)2SO4) and 1.5 U Taq DNA polymerase.The DNA was amplified using the following thermal cycling steps: 94℃ for 3 min; 13 cycles at 94℃ (30 s), 45℃ (45 s), 72℃ (45 s) and 72℃ for 1 min.The PCR1 products were diluted 100 times, 2 μL of which was used as the template for PCR2 using the same primers and the same thermal cycling steps used above for PCR1.The DNA product produced by PCR2 was concentrated by standard ethanol precipitation and sent out for deep sequencing using an Illumina Miseq system at Sangon Biotech Company.

RT-PCR-based pull-down assay
Each aptamer candidate was incubated with IL-6-coated CAMBs or sIL-6R-coated CAMBs in 1× binding buffer.After incubation for 1 h, the tube was placed on a magnetic test tube holder and the supernatant was removed.After that, the beads were washed three times with 1× binding buffer.Protein-bound aptamers were eluted with ultrapure water at 95℃ for 15 min.Real-time PCR was used to quantify the eluted aptamers and the reaction conditions were using the following parameters: 30 cycles of denaturation at 94°C for 2 min, annealing at 45°C for 45 s, and elongation at 72°C for 45 s.The apparent dissociation constants (Kd) of all aptamer candidates were obtained by fitting the fluorescence value of the bound fraction of aptamers by varying total aptamer concentration, while the total target concentration is fixed.y = D + (A -D)/(1 + (K/x) B ), where y = fluorescent intensity, x = aptamer concentration, A = maximum fluorescent intensity, B = the slope factor, K = Kd and D = minimal fluorescent intensity.

Microscale thermophoresis (MST)
IL-6 and sIL-6R proteins were labelled using Monolith RED-HNS second generation protein labelling kit.MST binding experiments were carried out using a constant concentration of labelled IL-6 and sIL-6R in 1 × binding buffer and varying concentrations of the aptamer.The experiments were carried out on a Monolith NT.115 Pico (NanoTemper Technologies, Munich, Germany) at 25℃ using standard capillaries.The recorded fluorescence was normalized and fitted using the Kd fit formula.Duplicate measurements were performed for each experimental setup.y = D + (A -D)/(1 + (K/x) B ), where y = fluorescent intensity, x = aptamer concentration, A = maximum fluorescent intensity, B = the slope factor, K = Kd and D = minimal fluorescent intensity.

Prevention assay
In the IL-6 binding prevention assay, 200 nM CIL-6A6-1 was incubated with 200 nM sIL-6R-coated CAMBs at RT for 1 h so that CIL-6A6-1 would bind sIL-6R-coated CAMBs.The beads were washed twice using 1× binding buffer and resuspended in either 30 μL of 1× binding buffer containing 200 nM IL-6 (+target) or 30 μL of 1× binding buffer (-target).After incubation for 1 h, the beads were washed twice and the amount of CIL-6A6-1 remained on beads (A+target and A-target) was quantified using the realtime-PCR-based pull-down assay described above.To correct for non-specific adsorption by beads, CIL-6A6-1 incubated with bare CAMBs was included to derive the amount of CIL-6A6-1 that was nonspecifically adsorbed by beads (Abackground) The prevention efficiency (PE) is calculated as: PEIL-6P = [(A+target -Abackground) / (A-target -Abackground)]  100%.The PEIL-6P value was also derived for the control sequence (CRandom) using the same method.Three independent replicates were done for each sequence.The sIL-6R binding prevention assay was performed identically except for the use of IL-6-coated CAMBs and free sIL-6R.The prevention efficiency (PE) is calculated as: PEsIL-6RP = [(A+target -Abackground) / (A-target -Abackground)]  100%.The PEsIL-6RP value was also derived for CRandom.

Competition assay
In the IL-6 binding competition assay, 200 nM CIL-6A6-1 was incubated with 200 nM sIL-6R-coated CAMBs in either 30 μL of 1× binding buffer containing 200 nM IL-6 (+target) or 30 μL of 1× binding buffer (-target) at RT for 1 h so that CIL-6A6-1 would bind sIL-6R-coated CAMBs with or without the competition of IL-6.After incubation for 1 h, the beads were washed twice and the amount of CIL-6A6-1 remained on beads (A+target and A-target) was quantified using the realtime-PCR-based pull-down assay described above.To correct for non-specific adsorption by beads, CIL-6A6-1 incubated with bare CAMBs was included to derive the amount of CIL-6A6-1 that was nonspecifically adsorbed by beads (A background ).The competition efficiency (CE) is calculated as: CEIL-6P = [(A+target -Abackground) / (A-target -Abackground)]  100%.The CEIL-6P value was also derived for the control sequence (CRandom) using the same method.Three independent replicates were done for each sequence.The sIL-6R binding competition assay was performed identically except for the use of IL-6-coated CAMBs and free sIL-6R.The competition efficiency (CE) is calculated as: CEsIL-6RP = [(A+target -Abackground) / (A-target -Abackground)]  100%.The CEsIL-6RP value was also derived for CRandom.

Substitution assay
In the IL-6 binding substitution assay, 200 nM IL-6 was incubated with 200 nM sIL-6R-coated CAMBs at RT for 1 h.The beads were washed twice using 1× binding buffer and resuspended in 30 μL of 1× binding buffer containing 200 nM CIL-6A6-1 so that CIL-6A6-1 would bind sIL-6R-coated CAMBs and substitute IL-6 from the IL-6/sIL-6R complex.After incubation for 1 h, the beads were washed twice and the amount of CIL-6A6-1 remained on beads (A) was quantified using the realtime-PCR-based pull-down assay described above.To correct for non-specific adsorption by beads, CIL-6A6-1 incubated with bare CAMBs was included to derive the amount of CIL-6A6-1 that was nonspecifically adsorbed by beads (Abackground).Equal amount of CIL-6A6-1 originally added was also quantified by realtime-PCR (Asum).The substitution efficiency (SE) is calculated as: SEIL-6P = [(A -Abackground) / (Asum)]  100%.The SEIL-6P value was also derived for the control sequence (CRandom) using the same method.Three independent replicates were done for each sequence.The sIL-6R binding substitution assay was performed identically except for the use of IL-6-coated CAMBs and free sIL-6R.The substitution efficiency (SE) is calculated as: SEsIL-6RP = [(A -Abackground) / (Asum)]  100%.The SEsIL-6RP value was also derived for CRandom.
In the other binding substitution assay, 200 nM fluorophore labelled IL-6 was incubated with 200 nM sIL-6R-coated CAMBs at RT for 1 h.The beads are washed twice and resuspended in 1× binding buffer containing 2 μM CIL-6A6-1.After incubation for 1 h, the beads were washed twice and the amount of IL-6 remained on beads (F) was quantified using the fluorescence spectrum analysis.To correct for non-specific adsorption by beads, the bare beads incubated with fluorophore labelled IL-6 were included as the background signal (Fbackground).Equal amount of IL-6 originally added was also quantified by fluorescence spectrum analysis (Fsum).The substitution efficiency (SE) is calculated as: SEIL-6P = [1-(F -Fbackground) / (Fsum)]  100%.The SEIL-6P value was also derived for the control sequence (CRandom) using the same method.Three independent replicates were done for each sequence.The sIL-6R binding substitution assay was performed identically except for the use of IL-6-coated CAMBs and free sIL-6R.The substitution efficiency (SE) is calculated as: SEsIL-6RP = [1-(F -Fbackground) / (Fsum)]  100%.The SEsIL-6RP value was also derived for CRandom.

Stability assay
6 μM circular anti-human IL-6 aptamer and the corresponding linear aptamer were dissolved in 1× binding buffer and incubated in 50% human serum at 37℃ for 0, 2, 4, 8, 12, 24, 36, 48h.At established time points, 20 μL samples were heated at 95°C for 10 min to denature enzymes and then stored at -20°C until all samples were collected.Prior to electrophoresis analysis, samples were prepared for electrophoresis by addition of 2 × denaturing gel loading buffer to 1× final concentration.Then, samples were loaded and 10% 1× TBE denaturing PAGE was run at 110 V for 30 min at 25°C.The gel was visualized using a TOMOS Molecular Imager Gel Doc EX System and the bands were quantified using ImageJ software.The half-life value was determined by quantifying the time required for the aptamer concentration to degrade to 50% of its initial level.
RNA extraction, cDNA synthesis, and real-time quantitative PCR assay RAW264.7 cells were seeded in a 24-well culture plate at 10 5 cells / well and incubated overnight at 37℃ with 5% CO2.Cells were activated with 1 μg/ml LPS and simultaneously treated with various concentration of IL-6A6-1 circular aptamer (CIL-6A6-1) (2 μg/ml, 200 ng/mL, 20 ng/mL), 200 ng/mL CIL-6A, CIL-6A4-1, CIL-6A6-1 and CIL-6A46 or some other positive control groups (2 μg/ml sIL-6R-specific RNA aptamer AIR-3A, IL-6-specific aptamer IL62, random circular aptamer CRandom and Tocilizumab) for 12 h.For total RNA extraction, the adherent cells were lysed with Trizol reagent (Sangon Biotech, Shanghai, China).RNA from each sample was converted to cDNA by Takara PrimeScript RT reagent Kit (Takara, Dalian, China) according to the manufacturer's instructions.The qPCR reaction was performed by Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA) and the reaction conditions were using the following parameters: 30 cycles of reactions of denaturation at 95°C for 2 min, annealing at 55°C for 30 s and elongation at 72°C for 45 s.The expression of target genes, including IL-6, sIL-6, IL-1β, and COX-2 were evaluated in this study.Primer sets of target genes and the actin gene (β-Actin) for qPCR are shown in Supplementary Table S3.

Flow cytometry
RAW264.7 cells were seeded in a 6-well culture plate at 10 6 cells / well and incubated overnight at 37℃ with 5% CO2.After incubation, cells were activated with 1 μg/ml LPS and simultaneously treated with 2 μg/ml of IL-6A6-1 circular aptamer and some other positive control groups (2 μg/ml sIL-6R-specific RNA aptamer AIR-3A, IL-6-specific aptamer IL62, CRandom and Tocilizumab) for 12 h.At the indicated time, the anti-CD16/32 antibody (Biolegend 101319, California, United States) blocked the Fc receptor on the surface of RAW264.7 cells and incubated on ice for 10 min.Then, macrophages were harvested and stained with the fluorescein diacetate (FDA, live cells stain) and APC-conjugated anti-mouse CD80 in the dark for 30 min at 4°C.After that, the labeled cells were analyzed using a flow cytometer (Beckman Coulter Navios, San Jose, CA, USA).Data were analyzed with Flow Jo 10 software.

Fluorescence measurements of G-quadruplex-ligand interaction
Thioflavin T (ThT, 10 μM) and the relevant circular DNA (300 nM) were incubated in the 1× binding buffer for 30 min.Fluorescence measurements were carried out in a 96-well assay plate using a microplate reader (THERMO Varioskan Flash) at room temperature.The excitation wavelength of the solution was set at 425 nm, and the emission spectra were collected from 450 to 600nm with a step of 2 nm.N-methylmesoporphyrin Ⅸ (NMM, 5 μM) and the relevant circular DNA (300 nM) were incubated in the 1× binding buffer for 30 min.Fluorescence measurements were carried out in a 96-well assay plate using a microplate reader (THERMO Varioskan Flash) at room temperature.The excitation wavelength of the solution was set at 399 nm, and the emission spectra were collected from 550 to 780nm with a step of 2 nm.

Circular dichroism analysis
Circular dichroism (CD) studies were carried out using JASCO J-1700 instrument according to the manufacturer's instructions.All constructs were scanned from 320 to 220 nm at 16 μM DNA in a 0.1-cm quartz cuvette.All DNA samples were incubated for 30 min at room temperature in the 1× binding buffer before scanning.Table S1.

Figure S5 .
Figure S5.Assessment of RT-PCR-based efficiency of CIL-6A6-1 towards interfering the interaction between IL-6 or sIL-6R in three different assays: a) prevention assay, b) competition assay and c) substitution assay.CRandom sequence was used as control.

Figure S8 .
Figure S8.Fluorescence emission spectra of N-methylmesoporphyrin Ⅸ (NMM) in the presence of CIL-6A6-1 at different Li + concentrations.The excitation wavelength was set at 399 nm.

Table S2 .
The Kd values of AIR-3A and its DNA counterpart against sIL-6R measured using MST

Table S3 .
Primer for RT PCR used in this work