Encoding function into polypeptide-oligonucleotide precision biopolymers

We report a novel synthesis strategy to prepare precision polymers providing exact chain lengths, molecular weights and monomer sequences that allow post modifications by convenient DNA hybridization.


Material and methods
Copolymer dHSA-(ssDNA)2 preparation and purification 1 mg native HSA was dissolved in degassed urea buffer (2 mL, 5 M urea, 50 mM phosphate buffer, 2 mM EDTA, pH 7.4) and stirred at room temperature for 15 min. TCEP (0.5 mg, 100 equivalent) was added as solid and the reaction mixture was stirred for additional 30 min under argon atmosphere. Then the maleimide terminated ssDNA (2.8 mg dissolved in 0.28 mL milli-Q water, 40 equivalent, synthesized,

Maleimide-ssDNA
The reaction mixture was purified by ultrafiltration (30 KD molecular weight cut off) 5 times with urea buffer and 3 times with milli-Q water to remove the unreacted DNA and small molecules. Then the DNA stabilized dHSA was diluted to 1 mL in milli-Q water and loaded onto an anion exchange column (GE healthcare MonoQ 4.6/100 PE) mounted to fast protein purification system (GE healthcare Äkta purifier 10) (Buffer A 50 mM phosphate buffer, Buffer B 50 mM phosphate 1 M NaCl) to separate the macromolecules from the statistically protein-DNA conjugation mixture. All the seven fractions were collected and washed by ultrafiltration for 5 times to remove the salt, and preserved in 100 µL Mille-Q water. The 7 Fractions could be observed in the chromatogram (F1-7, Figure S2a). All fractions were analysed by SDS-PAGE gel ( Figure S2b). F7 could not be stained with coomassie blue and was therefore attributed to free DNA. All other fraction could be stained and where analysed with ImageJ software. The following ratios were obtained for each fraction: F1: 5.5%, F2: 1.7%, F3: 5.3%, F4: 49.0%, F5: 24.5%, F6: 14.1. F1-3 was discarded because they only contain minor amount of sample. F 4-6 were analysed by MALDI-TOF. Only F4 showed a single peak, in contrast to F5, which showed a mixture of different compounds ( Figure S2c and d), and F6, which could not be detected during the MLDI-TOF measurement at all. Therefore, F4 was identified as main product and used for all following experiments. F4 contained two bound DNA molecules as determined from the MALDI-TOF spectrum and was therefore referred to as dHSA-(ssDNA)2. In the denatured SDS-PAGE ( Figure S2b), the shift band of the copolymer is near 72KD compared to the protein maker, which is different from the MALDI-TOF result. The reason was attributed to the enhanced negative charge from the attached oligonucleotides, although the copolymers was denatured with SDS, the charge has still an effect on shift distance in the SDS-PAGE.

MLDI-TOF measurement of the copolymers
All MALDI measurements were performed with a Reflex III equipped with a nitrogen laser (Bruker Daltonics). The machine was operated by flex control and the data was analysed by flex analysis.
The molecule weight of SST-SH ( Figure S6b  The molecule weight of copolymer fraction 4 and 5( Figure S2c and d) was measured by MALDI-TOF.

Transmissions electron microscope imaging
A Jeol 1400 Transmissions electron microscope was used to measure the bright field TEM images of dHSA-(ssDNA)2 ( Figure 1d). The sample was prepared by dropping dHSA-(ssDNA)2 (10 µg/mL, 5 µL dissolved in milli-Q water) on a freshly glow discharged 300 mesh size copper grid covered with a continuous carbon film and dried overnight at room temperature. Then the copper grid was stained by uranyl acetate.
Dynamic light scattering size and zeta potential measurement The DLS size and zeta potential of dHSA-(ssDNA)2 ( Figure S3a and S3d, sample was dissolved in 10 mM KCl buffer, 100 µg/mL, 600 µL) was measured by a Malvern Zeta sizer ZEN3600 (Malvern Ltd, Malvern, UK) at 25°C with 173° angle.

Circular dichroism (CD) measurement
Circular dichroism was in measured to identify the change of molecular structure of the copolymer dHSA-(ssDNA)2 ( Figure S3b and c). As observed from the CD spectra, an increase in ordered α-helix and a decrease in β-sheet, β-turn and random coil elements was observed for the dHSA-(ssDNA)2 copolymer in comparison to native HSA or dHSA-PEO as published before. 2 Denatured HSA (dHSA) could not be used as control as it precipitates from solution without the stabilizing side chains.    Figure S5b). The GFP-SH and maleimide-ssDNA* conjugation as well as the purified GFP-ssDNA* was monitored by 10% native PAGE ( Figure S5a). The isolated original GFP-SH as well as the purified GFP-ssDNA* were measured by MALDI-TOF to identify the mass (Figure 1e). The purified GFP-ssDNA* was stored in the purification condition (50 mM phosphate with 0.45 M NaCl, 55% of Buffer A and 45% of Buffer B).
The SST-SH was conjugated to maleimide-ssDNA* to generate SST-ssDNA* (Figure 1g). The SST-SH with maleimide-ssDNA* (1 and 2 equimolar ratio) were mixed in 10 mM MgCl2 buffer, 10% Methanol and incubated for 4 h on shaker at room temperature (400 RPM). The outcome of the reaction, especially unreacted ssDNA* was determined by native PAGE with aluminum sulfate containing Coomassie Brilliant Blue according to a previously published protocol. 5 The conjugation efficiency of SST-ssDNA* was tested with native PAGE ( Figure S6c) and the reaction mixture was purified through anion exchange column (GE healthcare MonoQ 4.6/100 PE) mounted on a fast protein purification system (GE healthcare Äkta purifier 10) (Buffer A 50 mM phosphate buffer, Buffer B 50 mM phosphate 1 M NaCl) (UV absorbance spectra in Figure S6d). The formed SST-ssDNA* (MALDI-TOF spectra in Figure S7) with MW 6701 Da was isolated and loaded to copolymer dHSA-(ssDNA)2 by DNA hybridization.      Y-shaped DNA* linker (YDNA*) was constructed by ssDNA S1, S2, S3 with half of the sequence complementary to each other. S1 was equipped with a sticky end, complementary to the ssDNA from the copolymer dHSA-(ssDNA)2. S2 and S3 was labelled with chromophore Atto594 and Atto655 as modal functionalities, separately. The YDNA was constructed by ssDNA S1, S2 and S3 hybridization in 1× TAE buffer (S1 : S2 : S3 = 1.2 : 1 : 1). The reaction proceeded with high conjugation efficiency ( Figure   S11) and the YDNA was loaded to the copolymer dHSA-(ssDNA)2 without any further purification.