Exploiting the Protein Corona from Cell Lysate on DNA Functionalized

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Exploiting the Protein Corona from Cell Lysate on DNA Functionalized Gold Nanoparticles for Enhanced mRNA Translation Kian Ping Chan, Yang Gao, Jeremy Xianwei Goh, Dewi Susanti, Eugenia Li Ling Yeo, Sheng-Hao Chao, and James Chen Yong Kah ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15269 • Publication Date (Web): 09 Mar 2017 Downloaded from http://pubs.acs.org on March 9, 2017

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ACS Applied Materials & Interfaces

Exploiting the Protein Corona from Cell Lysate on DNA Functionalized Gold Nanoparticles for Enhanced mRNA Translation Kian Ping Chan1,2,3, Yang Gao1, Jeremy Xianwei Goh1, Dewi Susanti4, Eugenia Li Ling Yeo1, Sheng-Hao Chao*2,5, James Chen Yong Kah*1,3 1

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Department of Biomedical Engineering, National University of Singapore, Singapore

Bioprocessing Technology Institute, Agency for Science, Technology and Research, Singapore 3

NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore 4

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Faculty of Science, National University of Singapore, Singapore

Department of Microbiology and Immunology, National University of Singapore, Block MD4, 5 Science Drive 2, Singapore 117597

KEYWORDS Gold Nanoparticles, DNA, non-specific adsorption, protein corona, hybridization, mRNA translation

ACS Paragon Plus Environment

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ABSTRACT

This study describes the use of DNA functionalized gold nanoparticles (AuNPs) to enhance the synthesis of proteins in cell lysate and examines the mechanisms behind the enhanced mRNA translation. With an appropriate DNA oligomer sequence that hybridizes to the 3’-untranslated region of two mRNA of interest: insulin and green fluorescent protein (GFP), we found that these DNA conjugated AuNPs (AuNP-DNA) introduced into HeLa cell lysate enhanced the synthesis of insulin and GFP by up to 2.18 and 1.80-fold respectively over baseline production with just the mRNA present. The insulin synthesis was markedly reduced with non-DNA citratecapped AuNP (1.25-fold), and AuNP-DNA with a non-specific poly(T) sequence (1.25-fold). We showed that both non-specific adsorption of ribosomes and translation factors to form a lysate protein corona on AuNP-DNA and weak hybridization between DNA oligomers and mRNA of interest were important factors that brought translation factors, ribosomes and mRNA into close proximity of each other. This could reduce the recycling time of ribosomes during mRNA translation, thereby increasing the efficiency of protein synthesis. The outcome of this work shows that with rational DNA design, it could be possible to modulate intracellular biological processes with AuNP-DNA and increase their production of proteins for various biomedical applications.


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INTRODUCTION The regulation of mRNA translation into proteins is an important cellular process where dysregulation is intimately associated with a range of pathological conditions, notably cancer1, neurological disorders2, and diabetes which is caused by inability of beta cells to synthesize insulin3. Protein synthesis is a complex process mediated by ribosomes and it involves the recruitment of mRNA, tRNA, amino acids and numerous other proteins known as translation factors (eukaryotic initiation (eIF), elongation (eEF) and release factors (eRF) and poly(A)binding protein (PABP) etc.), to form a coordinated complex that allows the ribosomes to translate the mRNA into the protein of interest4-5. DNA functionalized nanoparticles (NP-DNA) has been shown to modulate protein synthesis. While Park et al. showed enhancement of in vitro translation by NP-DNA6-7, most NP-DNA were designed to inhibit protein synthesis, either by conjugating oligonucleotides complementary to the mRNA of interest8 or siRNA9-11. These conjugated oligonucleotides exerted their functionality upon triggered release from the NPs via an external energy source or otherwise. The photothermal property of gold nanoparticles (AuNPs) makes them an ideal candidate for facilitating triggered release of bound DNA by optically exciting the AuNPs at their surface plasmon resonance (SPR) to induce localized heating to release the DNA using a laser12-13. AuNPs also allow easy conjugation to thiol-terminated DNA14-16, and the resulting DNA conjugated AuNPs (AuNP-DNA) exhibit efficient cell uptake8 and colloidal stability in biological environment15, 17. However, AuNPs suffer from non-specific adsorption of proteins on their surface to form a protein corona when introduced into biological media6, 18-23. The surface masking by protein


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corona has shown to adversely affect the intended functionality of NPs24-26, leading many to regard the non-specific protein adsorption as undesirable. Fortunately, the inevitable protein corona on AuNPs could also be exploited for useful applications in drug delivery12, 27-28, biosensing29 and assay development30. Here, we propose an approach to enhance protein synthesis using AuNP-DNA which initiates self-assembly of mRNA translation machinery through non-specific adsorption of translation factors and ribosomes found in lysate to form a lysate protein corona on AuNP-DNA. We hypothesize that the close proximity between the translation factors, ribosomes and mRNA of interest facilitated by AuNPDNA will reduce the recycling time of ribosomes during mRNA translation (Scheme 1), thereby increasing the efficiency of protein synthesis. In this way, protein synthesis can be enhanced without triggered DNA release.


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Scheme 1. Enhancement of mRNA translation using AuNP-DNA and mRNA of interest (blue lines). (a) In the absence of AuNP-DNA, mRNA templates are dispersed in biological media and the recycling of ribosomes is slower compared to (b) mRNA translation enhancement using AuNP-DNA where a higher density of mRNA templates hybridized on AuNP-DNA allows quick recycling of ribosomes. (i) Hybridization between 3’-untranslated region (UTR) of mRNA and DNA oligomer brings mRNA close to ribosomes and translation factors on AuNP-DNA. (ii) Assembly of small and large ribosomal subunits at the 5’-UTR of mRNA recruited by the translation factors in the lysate protein corona of AuNP-DNA for the initiation of translation. (iii) Elongation of translation. (iv) Termination of translation disassembles the large and small ribosomes near the AuNP-DNA, which (v) quickly reassembles onto the next nearby mRNA template for the next round of mRNA translation.

In this study, we extend the work of Park et al. by using an alternative site of DNA oligomer hybridization to the 3’-untranslated region (UTR) of mRNA that does not affect the recruitment of ribosomes at 5’-UTR. We also elucidate the mechanism and criteria for the enhanced mRNA translation and demonstrated application in a disease relevant protein, insulin closely related to diabetes mellitus. We showed that translation factors and ribosomal proteins were recruited to form a lysate protein corona on AuNP-DNA; and with appropriate oligomer sequence that hybridized to the mRNA of interest, we demonstrated 2.18-fold and 1.80-fold specific enhancement of insulin and green fluorescent protein (GFP) mRNA translation in vitro respectively. Complementary hybridization between DNA oligomers and mRNA was necessary for translation enhancement, but yet had to be sufficiently weak for maximum enhancement. The understanding of mechanism behind translation enhancement by AuNP-DNA gained from this


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study allows us to establish guidelines for rational design of DNA oligomer sequences, eventually leading to better design of the AuNP-DNA construct for optimized and specific protein synthesis.

MATERIALS AND METHODS Synthesis and characterization of AuNPs AuNPs were synthesized based on a previously established protocol31. Briefly, 15 mL of 34 mM trisodium citrate (Na3C6H5O7) was added to a boiling 1 mM hydrochloroauric acid (HAuCl4) under vigorous stirring for 15 min. The synthesized citrate-capped AuNPs were then cooled to room temperature and purified by repeated centrifugation at 9,000 rpm for 20 min with nuclease free water before use. The optical properties of AuNPs from UV-Vis absorption spectroscopy (UV-2450, Shimadzu, Japan) was used to derive their concentration32. The zeta potential (ζ) and hydrodynamic diameter (DH) of NPs from dynamic light scattering (DLS) were measured using a Zetasizer (Nano ZS, Malvern, UK) at 25°C. The AuNP morphology was examined under transmission electron microscopy (TEM) (JEM-1220, JEOL Ltd., Japan).

Selection of DNA oligomer sequences DNA oligomers (Integrated DNA Technologies, USA) were designed to include a 5’-thiol end modification for covalent thiolate-gold binding to the AuNP surface, followed by a 10 thymine bases poly-T spacer to minimize self-adsorption of DNA on the AuNP surface33, before the hybridization sequence of interest at the 3’ end (Figure 1). The sequence of interest was selected to hybridize to the 3’-untranslated region (UTR) downstream of the insulin coding sequence of a hemagglutinin (HA) tagged insulin (INS-HA) plasmid whose nucleotide was sequenced by Axil


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Scientific Pte. Ltd., Singapore. The 3’-UTR region was shown to be instrumental in mRNA translation34.

Figure 1. Design of DNA oligomer sequences for insulin mRNA. ctDNA is a control sequence with 15 bases of thymine at 3’-end. wkDNA has weak binding to 3’-UTR region of INS-HA mRNA while stDNA has strong binding to 3’-UTR region of INS-HA mRNA. All three DNA oligomers have 10 bases of thymine as spacer followed by a thiol terminal group at the 5’-end of the sequence of interest for thiolate bonding on the surface of AuNP.

Three DNA oligomers were used in this study to investigate the dependence of translation enhancement on the binding affinity between DNA oligomers and insulin mRNA (Figure 1): (1) a nucleotide sequence that exhibit weak affinity to 3’-UTR of insulin mRNA (wkDNA), (2) another nucleotide sequence that exhibit strong affinity to 3’-UTR of insulin mRNA (stDNA), and (3) a negative control sequence comprising of 15 thymine nucleotides only (ctDNA) that does not bind to the 3’-UTR.


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In selecting the DNA sequence, the 3’-UTR region of mRNA was divided into blocks of 15 bases and blocks with complementary base-pairing that would self-hybridize to the mRNA of interest were eliminated. We then determined the binding affinity of DNA oligomers with the remaining blocks in the mRNA after taking into account all possible secondary structures of the insulin mRNA and selected the one with the desired binding energies. The binding strength of DNA oligomers with the 3’-UTR of insulin mRNA was predicted by first using Sfold 2.2 to predict the range of possible secondary structures of insulin mRNA (see Supporting Information, Figure S1), followed by determining the number of base-pairing between DNA oligomers and the non-self-hybridized loop region of the mRNA available for binding to the DNA. Finally, the overall binding energy was determined from the known binding energy of individual A-T and G-C base pairs35. The wkDNA and stDNA oligomers were selected to avoid complementary base pairing in the insulin coding region, thereby reducing the disruption in INS-HA mRNA translation through non-specific hybridization. The location of hybridization between the different sequences and insulin mRNA is shown in Figure 1. To demonstrate specificity of the DNA oligomers towards translation of a specific mRNA, another set of DNA oligomers (gfpDNA and ctDNA as control) was used to show enhancement of GFP mRNA translation (Figure 2). The same approach was used to design the DNA oligomer that was predicted by Sfold 2.2 to exhibit weak binding to GFP mRNA (data not shown).


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Figure 2. Design of DNA oligomer sequence for GFP mRNA. gfpDNA has weak binding to GFP mRNA at the 3’-end. Both DNA oligomers have 10 bases of thymine as spacer followed by a thiol terminal group at the 5’-end of the sequence of interest for thiolate bonding on the surface of AuNP.

AuNP-DNA conjugation When received, the thiol terminal group of the DNA oligomers was protected with a mercaptopropanol disulphide bond. Prior to conjugation, the disulphide bond was reduced by tris (2-carboxyethyl) phosphine (TCEP) (Sigma Aldrich, USA) to create single stranded DNA oligomers by incubating a mixture of 1 µL of 100 µM DNA, 10 µL of 40 mM TCEP and 89 µL of 1x Tris-borate-EDTA (TBE) buffer for 3 h at room temperature according to the recommended protocol from the manufacturer. Unlike dithiothreitol (DTT) which was commonly used to reduce the protected thiol groups, TCEP removal was not required as the excess TCEP would not interfere with the conjugation process. Conjugation of different ratios of DNA oligomers to AuNPs was performed as previously reported15, 36 to determine the maximum


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density of DNA oligomers on AuNPs. For an incubation ratio of 120 DNA molecules per AuNP, 120 µL of 1 µM DNA was added to 100 µL of 10 nM AuNPs before 55 µL of citrate buffer (100 mM Na3C6H5O7 and 242 mM HCl) was added to the mixture under continuous vortexing. The AuNP-DNA conjugates were left to stand for 5 min before repeated centrifugation at 9,000 rpm for 20 min with nuclease free water to remove excess unbound DNA oligomers and TCEP from the AuNP-DNA. To quantify the bound DNA oligomers, the amount of excess unbound DNA oligomers in the supernatant during the two centrifugal washes was quantified by fluorescence of SYBR Gold nucleic acid stain (Life Technologies, US) against calibrating standards of known DNA concentrations using Infinite® 200 PRO (Tecan, Switzerland) and subtracted from the initial amount of DNA added. The optical properties, ζ and DH of AuNP-DNA were characterized in the same manner as the synthesized citrate-capped AuNPs.

Transcription of human insulin DNA plasmid in vitro Human influenza HA tag was incorporated into human insulin DNA plasmid (Origene Technologies, US) to facilitate subsequent detection of translated insulin with Western Blot. The HA-tagged human insulin DNA plasmid was linearized before adding to MEGAscript® T7 Transcription Kit (Life Technologies, US) for transcription in vitro to produce uncapped preproinsulin mRNA (INS-HA mRNA). Purification of INS-HA mRNA was performed using the MEGAclear™ Transcription Clean-Up Kit (Life Technologies, US). The concentration of INS-HA mRNA was measured using NanoDrop 2000c (Thermo Fisher Scientific, USA) before storage at -20 ºC. Transcription was similarly performed for GFP plasmid to generate GFP mRNA.


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Translation of preproinsulin mRNA in vitro Synthesis of preproinsulin protein, a precursor of insulin, from INS-HA mRNA was performed using a 1-Step Human In Vitro Protein Expression Kit (Human IVT kit, Thermo Scientific, USA). In each reaction, 1 µg of INS-HA mRNA and 12 µL of 10 nM AuNP or AuNP-DNA was added to 25 µL of HeLa cell lysate from the kit and incubated for 2.5 h at 37 oC. As a control, 1 µg of GFP mRNA was added in place of INS-HA mRNA to probe for the specificity of the AuNP-DNA towards INS-HA mRNA instead of GFP mRNA. The same was probed vice versa to study the specificity of AuNP-gfpDNA towards GFP mRNA with INS-HA mRNA as a control. The preproinsulin synthesized was the precursor form of insulin prior to post-translational modification. Therefore, we subsequently refer to it as insulin in the rest of the manuscript for simplicity. To determine concentration-dependence in mRNA translation, a range of AuNP or AuNP-DNA concentrations: 1.25, 2.5, 5, 10, 20 and 30 nM were introduced to the HeLa cell lysate.

Quantification of synthesized proteins in vitro The synthesized insulin and GFP were quantified using western blot. Anti-HA antibody (H3663, Sigma, 1:2000) and anti-GFP antibody (sc9996, Santa Cruz Biotechnology, Inc., 1:500) were used as primary antibodies to probe for INS-HA and GFP respectively. Anti-actin (Millipore, 1:2000) was used to detect actin, an abundant protein found in cell, for normalizing against variations in sample loading in the protein quantification step. Horseradish peroxidase (Pierce, US) conjugated secondary antibodies were used for detection. After antibody incubation,


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polyvinylidene difluoride (PVDF) blots were washed with PBS-Tween (10 mM sodium phosphate, 0.15M NaCl, 0.05% Tween-20) and incubated with SuperSignal West Pico Substrate (Pierce, US). The intensity of protein bands was analyzed and quantified using ImageJ software. The amount of INS-HA and GFP were quantified after normalizing to actin as their respective loading control. Here, the baseline production level of INS-HA and GFP in the absence of AuNP-DNA was normalized to 1.00 as a reference. Triplicates were performed for all experiments, and statistical analyses for comparison between two groups were performed with one-tailed unpaired student’s t-test since an enhancement in translation was expected.

Non-specific protein adsorption on AuNP-DNA To demonstrate non-specific absorption of HeLa lysate proteins which include the translation factors around the AuNPs, agarose gel electrophoresis of AuNP-DNA before and after incubation in HeLa cell lysate was performed using 1x TBE buffer and a constant voltage of 100V for 1 h. The relative amount of proteins non-specifically adsorbed on AuNPs was probed by incubating AuNPs in HeLa lysate for 2.5 h at 30 oC to reproduce the conditions as the translation step. The HeLa lysate containing the AuNPs was centrifuged at 2,000 g for 10 min to remove nonadsorbed proteins. Here, low speed centrifugation was used to avoid separation of adsorbed proteins from the surface of AuNPs and repeated centrifugation was avoided to minimize proteins exchange in the washing buffer. A SDS-PAGE was performed by loading the pellet containing the recovered AuNP-DNA with lysate protein corona into Novex™ 4-12% TrisGlycine Mini Protein Gels (EC6038BOX) and running the samples at 110V for 120 min. Here,


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the AuNPs were trapped in the wells while the adsorbed proteins migrated into the gel, which was then stained with Bio-Safe™ Coomassie Stain (#1610786) to show the relative amount and size profile of proteins adsorbed on the AuNP-DNAs and AuNPs. We further proved the localization of ribosomes on the lysate protein corona which facilitated the translation enhancement, and determined the amount of ribosomal proteins adsorbed on the AuNPs using Western Blot. Anti-ribosomal protein S10 antibody (ab151550, 1:1000) and antiribosomal protein L26 antibody (ab5956, 1:1000) were used as primary antibodies to detect the presence of both small S10 and large L26 ribosomal sub-units protein respectively; anti-actin (Millipore, 1:2000) was used as a control as described previously. The same secondary antibodies conjugated to horseradish peroxidase as described above were used for labeling the ribosomal bands. The labeled bands were analysed using ImageJ and the amount of ribosomal proteins were quantified after normalization to the actin control.

Hybridization between DNA oligomers and mRNA To prove that hybridization between AuNP-DNA and mRNA was necessary for enhanced mRNA translation, we incubated the DNA/RNA hybridized duplex substrate with ribonuclease H (RNase H), a non-sequence-specific endonuclease that recognizes and catalyzes the cleavage of the 3’-O-P bond of RNA. This reduced the number of DNA-RNA complex between DNA oligomers and INS-HA mRNA and inhibited the translation enhancement. Western Blot was performed after RNase H treatment where 1 µL of RNase H (50U/ µL) was added to the translation mix and incubated for 2.5 h, before the amount of translated INS-HA mRNA was quantified as described previously.


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RESULTS AND DISCUSSION Characterization of DNA conjugated AuNPs The synthesized citrate-capped AuNPs were isolated and monodisperse, with an average diameter of 11.7 ± 0.09 nm as measured from TEM images (Figure 3a and b). The UV-Vis absorption spectrum showed a peak at 517 nm (Figure 3c). With DNA conjugation, we observed a slight redshift in the peak absorbance to 522 nm for all AuNP-DNAs, likely due to a change in the local refractive index on the AuNP surface from a citrate-capped to a DNA-conjugated surface37-38. Peak broadening was also not significant upon DNA conjugation, indicating that the AuNP-DNA maintained colloidal stability after conjugation.

Figure 3. Characterization of synthesized citrate-capped AuNPs and their conjugates with all three types of DNA oligomers (AuNP-DNA). (a) TEM image of the synthesized AuNPs. (b) Histogram of the size distribution of synthesized AuNPs determined from the TEM image. (c)


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UV-Vis spectra showed a defined peak of 517nm and 522nm for AuNPs and AuNP-DNA respectively. (d) Average hydrodynamic diameter, DH measured from dynamic light scattering (DLS) indicating the absence of large aggregates formation. (e) Zeta potential, ζ of all samples was negative due to negatively charged citrate capping or DNA. (f) Similar DNA density of about 80 DNA oligomers per AuNP was obtained for all three AuNP-DNAs.

The citrate-capped AuNPs had a mean DH of 15.85 ± 0.28 nm, which increased slightly with DNA conjugation (DH, AuNP-ctDNA = 22.77 ± 0.99 nm, DH, AuNP-wkDNA = 32.91 ± 2.19 nm, DH, NP-stDNA = 29.70 ± 0.40 nm and DH, NP-gfpDNA = 30.97 ± 2.41 nm) due to the presence of 25-mers DNA oligomers approximately 8.5 nm (Figure 3d). This confirmed the absence of large aggregates formation after DNA conjugation. The zeta potential of all AuNP-DNAs remained negative after conjugation since the phosphate backbone of DNA was also negatively charged (Figure 3e). With a DNA:AuNP incubation of 120:1, we were able to conjugate 77.5 ± 6.5, 77.0 ± 3.9, 81.5 ± 7.6 and 84.1 ± 2.6 DNA oligomers per AuNP for AuNP-ctDNA, AuNP-wkDNA, AuNP-stDNA and AuNP-gfpDNA respectively (Figure 3f), with no statistically significant difference between the four DNA oligomers (one-way ANOVA (F(3, 26) = 0.2393, p = 0.8681). The DNA density was comparable to those reported by others39.

Enhancement in insulin mRNA translation We chose INS-HA mRNA as the template for translation instead of direct protein synthesis from a coupled transcription-translation of INS-HA plasmid to avoid fluctuations in transcription efficiency. The introduction of AuNP, AuNP-ctDNA and AuNP-wkDNA into the HeLa lysate together with INS-HA mRNA resulted in an enhancement of protein synthesis by a factor of


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1.26, 1.26, and 2.18 respectively, relative to the baseline synthesis level of INS-HA in the absence of AuNPs (Figure 4).

Figure 4. Enhancement of INS-HA mRNA translation in vitro. (a) Western Blot results showing the qualitative amount of insulin synthesized with different AuNP-DNAs introduced into HeLa lysate with INS-HA mRNA, compared to a control without AuNPs. (b) The western blot results were quantitatively analyzed with ImageJ to show the enhanced synthesis of insulin with appropriate AuNP-wkDNA compared to citrate-capped AuNP and AuNP-ctDNA as controls. *One-tail t-test, p < 0.01, N = 10.


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In the absence of DNA oligomers, lysate proteins could also adsorb non-specifically on the citrate-capped AuNPs. This facilitated the recruitment of translation factors and ribosomal proteins close to each other, which enhanced the efficiency of INS-HA translation and hence an increase in translation enhancement by 1.26. This enhancement in translation was further enhanced by a factor of 2.18 with 10 nM of AuNP-wkDNA where the wkDNA oligomer was able to additionally recruit the INS-HA mRNA to the vicinity of the ribosomes and other translation factors through weak hybridization of the mRNA. This enhancement was also AuNPDNA concentration-dependent (see Supporting Information, Figure S2). With increasing concentration of AuNP-wkDNA up to 30 nM, we observed a corresponding increase in mRNA translation. It was plausible that a higher concentration of AuNP-wkDNA would provide more “focal points” for insulin mRNA and translational machinery proteins to assemble and concentrate, thus increasing the efficiency of translation. Such a strong enhancement was lost when the wkDNA was replaced by our control ctDNA oligomer which was unable to sufficiently hybridize and recruit the INS-HA mRNA. Here, the translation enhancement was 1.26, similar to citrate-capped AuNP.

Effect of DNA-mRNA binding affinity on translation enhancement The binding affinity in the complementary base pairing between DNA oligomers and mRNA also affected the mRNA translation enhancement. Here, the secondary structure of mRNA consisted of stem and loop regions (see Supporting Information, Figure S1) where base-pairing between mRNA and DNA is only possible at the loop region. We chose another DNA oligomer sequence (stDNA) that binds to another site in the 3’-UTR of the INS-HA mRNA with an


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average of 12.14 base-pairings and 30.01 hydrogen bonds in the loop region of all ten possible mRNA secondary structures identified (Figure S1), compared to wkDNA with 7.92 base-pairing and 22.46 hydrogen bonds. This led to a stronger binding energy between stDNA and INS-HA mRNA (Gibbs free energy, ∆G = -225.03 kcal/mol) compared to that between wkDNA and INSHA mRNA (-184.28 kcal/mol) (Figure 5a).

Figure 5. Weak binding between wkDNA and INS-HA mRNA resulted in higher mRNA translation enhancement than strong binding between stDNA and INS-HA mRNA. (a) Schematic diagram showing the two hybridization sites on 3’-UTR region of mRNA and affinity of the two DNA oligomers that bind respectively to these two sites. (b) Western Blot result provides a qualitative comparison of the INS-HA protein amount synthesized from AuNP-wkDNA and AuNP-stDNA. (c) Analysis of Western Blot using ImageJ showed that the translation of INS-HA mRNA was higher for AuNP-wkDNA compared to AuNP-stDNA, which did not significantly enhance the translation of insulin over control with mRNA only. *One tail t-test, p

Exploiting the Protein Corona from Cell Lysate on DNA Functionalized

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