Architecture of high-affinity unnatural-base DNA aptamers. toward pharmaceutical applications

Transcription

1 Supplementary Information rchitecture of high-affinity unnatural-base DN aptamers toward pharmaceutical applications Ken-ichiro Matsunaga 1,2,3, Michiko Kimoto 1,2,3,4, harlotte Hanson 5, Michael Sanford 5, Howard. Young 5,*, and Ichiro Hirao 1,2,3,* 1 Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, he Nanos, #04-01, Singapore , 2 agyx Biotechnologies, Suehiro-cho, surumi-ku, Yokohama, Kanagawa , Japan, 3 RIKEN enter for Life Science echnologies, Suehiro-cho, surumi-ku, Yokohama, Kanagawa , Japan, 4 PRESO, JS, Honcho, Kawaguchi-shi, Saitama , Japan, 5 Laboratory of Experimental Immunology, ancer and Inflammation Program, National ancer Institute at Frederick, Frederick, MD, US *orrespondence should be addressed to I. H. (ichiro@ibn.a-star.edu.sg) or H.. Y. (YoungHow@mail.nih.gov) 1

2 Supplementary able 1. Oligonucleotide sequences used in this study. he mini-hairpin DN sequence is red and underlined. he Ds positions and the substitutions from Ds to are shown in red. he pairs in aptamer 57mh that replaced the pairs in aptamer 57mh are shown in blue. Name ptamer 49 ptamer 49 ptamer 26 ptamer 58 ptamer 57mh ptamer 57mh Sequence 5 -DsDs Ds DsDs Ds-3 5 -Ds Ds-3 5 -Ds Ds-3 2

3 Only human serum Only human serum Only human serum ptamer 49 Single-stranded 50-mer DN1 Single-stranded 50-mer DN2 5 3 Ds Ds Ds ime (h) Original DN % % % recovery Supplementary Fig. 1. Stability analysis of aptamer 49 and control DNs in human serum. Each DN aptamer (5 l, aptamer 49, aptamer 58, aptamer 57mh, and aptamer 57mh; 50 M) or ontrol DN (5 l, ssdn1 and ssdn2: 50 μm) was mixed with human serum (120 l), and the mixture (2 M DN in 96% serum) was incubated at 37. liquots (10 l) were removed at various time points from 0 to 72 hours, and mixed with 110 l of denaturing solution (1 BE containing 10 M urea) immediately. Each sample was analyzed by 15% denaturing polyacrylamide gel electrophoresis. DN stained with SYBR old was detected with a bio-imaging analyzer (Fuji Film LS-4000) and quantified using the Multi auge software to determine the intact fraction. he analysis of each sample was repeated twice, and one of them is displayed in this figure. 3

4 Supplementary Fig. 2. hermal stabilities of DN aptamers and their derivatives. UV melting profiles of aptamer 49 (black, solid line), aptamer 49 (black, dotted line), aptamer 58 (blue, solid line), aptamer 57 (orange, solid line), and aptamer 57mh (red, solid line) were recorded, using a SHIMDZU UV-2450 spectrometer equipped with a temperature controller (MSP-8). he absorbance of each sample (2 μm in 1 mm KH 2 PO 4, 3 mm Na 2 HPO 4, and 155 mm Nal, ph 7.4) was monitored at 260 nm from 15 to 95, at a heating rate of 0.5 /min. Each melting temperature was calculated by the first derivative of the melting curve, using the IOR Pro software (WaveMetrics, Inc.). he melting profiles, normalized by the absorbance at 15 (left panel), and the first derivatives of the absorbance (right panel) are shown. 4

5 Residual amount (%) ptamer 57mh ptamer 57mh ptamer 58 ptamer 49 ssdn1 50-mer ssdn2 50-mer ime (h) Supplementary Fig. 3. Stability analysis of DN aptamer and control DN in human serum. he graph showed residual amount (%), aptamer 49 (N=4), aptamer 58 (N=2), aptamer 57mh (N=2), aptamer 57mh (N=2), ssdn1 50-mer and ssnd2 50-mer (N=2). Each DN aptamer (5 l, aptamer 49, aptamer 58, aptamer 57mh, and aptamer 57mh; 50 M) or ontrol DN (5 l, ssdn1 and ssdn2: 50 μm) was mixed with human serum (120 l), and the mixture (2 M DN in 96% serum) was incubated at 37. liquots (10 l) were removed at various time points from 0 to 72 hours, and mixed with 110 l of denaturing solution (1 BE containing 10 M urea) immediately. Each sample was analysed by 15% denaturing polyacrylamide gel electrophoresis. DN stained with SYBR old was detected with a bio-imaging analyzer (Fuji Film LS-4000) and quantified using the Multi auge software to determine the intact fraction. 5

6 Supplementary Fig. 4. SPR analysis of each DN aptamer variant against IFNγ in different running buffer and curve fitting with two types of binding model. he DN aptamer immobilization and monitoring of IFN-γ binding to DN aptamers were performed as described in the methods. Running buffer is 1 PBS (1 mm KH 2 PO 4, 3 mm Na 2 HPO 4 and 155 mm Nal, ph 7.4, ibco) supplemented with 0.05% (vol/vol) Nonidet P-40, in the presence or absence of the additional 50 mm Nal (205 mm or 155 mm Nal at the final concentration). he data were fitted with a 1:1 binding model (Equation 1) or heterogeneous ligand model (Equation 2) as indicated left on each sensorgram column, by using the BIevaluation 200 software, version 1.0 (E Healthcare). In heterogenous ligand model, the K D values of predominant tight binding were indicated as K D1, and minor binding (weak binding) as K D2. Fitting curves are plotted in black lines. he dissociation constants and chi square values, obtained from each curve fitting, were shown as the average with standard deviations with three data sets. 6

7 ptamer ng/ml ptamer ng/ml Unstimulated hlfng hlfng + aptamer ptamer ng/ml ptamer ng/ml ptamer ng/ml ptamer ng/ml ptamer 57mh 50 ng/ml ptamer 57mh 100 ng/ml ptamer 57mh 200 ng/ml ptamer 57mh 50 ng/ml ptamer 57mh 100 ng/ml ptamer 57mh 200 ng/ml Supplementary Fig. 5. bility of unnatural-base DN aptamers (50, 100, and 200 ng/ml) to neutralize human IFNγ (2 ng/ml) induction for S1 phosphorylation at 37, overnight. 7

8 Supplementary Fig. 6. bility of aptamer 57mh (200 ng/ml) to neutralize human IFNγ (2 ng/ml) induction for S1 phosphorylation over a 72-hour time course (10 min, 24 h, 48 h, and 72 h). 8