Inhibition of Xenograft Tumor Growth by Gold Nanoparticle-DNA Oligonucleotide Conjugates-Assisted Delivery of <i>BAX</i> mRNA

<div><p>Use of non-biological agents for mRNA delivery into living systems in order to induce heterologous expression of functional proteins may provide more advantages than the use of DNA and/or biological vectors for delivery. However, the low efficiency of mRNA delivery into live animals, using non-biological systems, has hampered the use of mRNA as a therapeutic molecule. Here, we show that gold nanoparticle-DNA oligonucleotide (AuNP-DNA) conjugates can serve as universal vehicles for more efficient delivery of mRNA into human cells, as well as into xenograft tumors generated in mice. Injections of <i>BAX</i> mRNA loaded on AuNP-DNA conjugates into xenograft tumors resulted in highly efficient mRNA delivery. The delivered mRNA directed the efficient production of biologically functional BAX protein, a pro-apoptotic factor, consequently inhibiting tumor growth. These results demonstrate that mRNA delivery by AuNP-DNA conjugates can serve as a new platform for the development of safe and efficient gene therapy.</p> </div

Influence of <i>BAX</i> mRNA delivery via AuNP-DNA conjugate or liposome on cell viability and mRNA half-life.

<p>(A) Relative viability of 5′BAX mRNA-transfected cells. HeLa cells were incubated with AuNP-αRNA I-5’BAX-null mRNA (Bax-null mRNA), AuNP-αRNA I-5’BAX mRNA, or liposome for 24 h. (B) RT-PCR analysis of 5′BAX mRNA to measure mRNA half-life. Amplification of cDNA from 5′BAX mRNA was achieved using a PCR primer (RNA I-AUG), designed specifically to bind to the 5’ UTR of 5′BAX mRNA, which is complementary to the cargo DNA of the AuNP-DNA. Equal loading was ensured using an internal control (GAPDH). The values were normalized against control cell values and are shown as the mean ± SEM (standard error of mean). Experiments were performed in triplicate and repeated at least three times.</p

Delivery of <i>dsRED</i> mRNA to HeLa cells by AuNP-DNA conjugates.

<p>(A) Measurement of the loading capacity of AuNP-αRNA I for 5′dsRED mRNA and 5′∆RNA I-dsRED mRNA. Known amounts of 5’ dsRED mRNA or 5′∆RNA I-dsRED mRNA (5, 10, and 20 pmol) and ten µl of varying concentrations (2, 4, and 8 µM) of these mRNAs hybridized to AuNP-DNA were loaded (lanes 4-6) to assess the loading capacity of for 5’ dsRED mRNA containing a sequence complementary to αRNA I at the 5′-terminus. The concentration of AuNP-DNA was 10 nM. Amounts of mRNA bound to AuNP-DNA at each concentration were plotted and shown in the graph. (B) Efficient delivery of AuNP-αRNA I-Cy3-5 ′dsRED mRNA into HeLa cells. Cells were treated with AuNP-αRNA I alone, AuNP-αRNA I-Cy3-5’∆RNA I-dsRED mRNA, or AuNP-αRNA I-Cy3-5’dsRED mRNA; these are represented schematically in diagrams. The final concentrations of AuNP-αRNA I and dsRED mRNA were 1 nM and 0.2 µM, respectively. Representative confocal fluorescence microscopy images of HeLa cells detecting Cy3-5’dsRED and Cy3-5’∆RNA I-dsRED mRNA (a) and dsRED protein (b) are shown. Transmission images of cells (c) and overlay images of a, b, and c (d) are also shown. Bar indicates 20 µm.</p

Retardation of tumor growth by delivery of AuNP-conjugated <i>BAX</i> mRNA.

<p>(A) The volume (mm³) of the tumor ((length × width² × π)/6) was determined over a five-week period after xenograft implantation followed by injection of 5 nM AuNP-αRNA I-5’BAX mRNA in mice. AuNP-αRNA I-5’BAX-null mRNA and liposome-5'BAX mRNA were used as controls. Data (<i>n</i> = 8) are presented as the mean ± SEM, and asterisks indicate statistically significant values as compared to the corresponding controls (^*<i>P</i> < 0.05). (B) Tumor weight was measured at the time of sacrifice (30 days after implantation) and is presented as the % of control. (C) Actual sizes of representative tumors are shown. (D) Effective delivery of Cy3-labeled 5′BAX mRNA by AuNP-DNA conjugates into tumor tissue. Green signals indicate Cy3-5’BAX mRNA. Bar indicates 200 µm. (E) Western blot analysis of the tissue lysates was performed using anti-BAX, anti-Caspase 3, and anti-β-actin antibodies.</p

Effects of directionality of mRNA binding to AuNP-DNA and presence of cap structure at the 5′-terminus of mRNA.

<p>HeLa cells were treated with AuNP-αRNA I hybridized to Cy3-5’dsRED mRNA, Cy3-3’dsRED mRNA, Cy3-5′, 3’dsRED mRNA, or Cy3-CAP-5’dsRED mRNA. Fluorescent images show the Cy3-labeled mRNA signal in the left panels and the red fluorescent signal of the dsRED protein in the right panels. Representative confocal microscopy images of HeLa cells for each treatment are shown, along with schematic representations of <i>in </i><i>vitro</i> transcripts and a diagram of the AuNP-αRNA I-mRNA binding structure. Bar indicates 20 µm. Relative intensities of fluorescence in cells were measured using ImageJ software. The values are shown by setting intensities of fluorescence from cells treated with AuNP-αRNA I + Cy3-5’dsRED mRNA as 100. Relative intensities of fluorescence were obtained from 10 cells of each treatment and are shown as the mean ± SEM (standard error of mean).</p

Modulation of RNase E Activity by Alternative RNA Binding Sites

<div><p>Endoribonuclease E (RNase E) affects the composition and balance of the RNA population in <i>Escherichia coli</i> via degradation and processing of RNAs. In this study, we investigated the regulatory effects of an RNA binding site between amino acid residues 25 and 36 (²⁴LYDLDIESPGHEQK³⁷) of RNase E. Tandem mass spectrometry analysis of the N-terminal catalytic domain of RNase E (N-Rne) that was UV crosslinked with a 5′-³²P-end-labeled, 13-nt oligoribonucleotide (p-BR13) containing the RNase E cleavage site of RNA I revealed that two amino acid residues, Y25 and Q36, were bound to the cytosine and adenine of BR13, respectively. Based on these results, the Y25A N-Rne mutant was constructed, and was found to be hypoactive in comparison to wild-type and hyperactive Q36R mutant proteins. Mass spectrometry analysis showed that Y25A and Q36R mutations abolished the RNA binding to the uncompetitive inhibition site of RNase E. The Y25A mutation increased the RNA binding to the multimer formation interface between amino acid residues 427 and 433 (⁴²⁷LIEEEALK⁴³³), whereas the Q36R mutation enhanced the RNA binding to the catalytic site of the enzyme (⁶⁵HGFLPL*K⁷¹). Electrophoretic mobility shift assays showed that the stable RNA-protein complex formation was positively correlated with the extent of RNA binding to the catalytic site and ribonucleolytic activity of the N-Rne proteins. These mutations exerted similar effects on the ribonucleolytic activity of the full-length RNase E <i>in vivo</i>. Our findings indicate that RNase E has two alternative RNA binding sites for modulating RNA binding to the catalytic site and the formation of a functional catalytic unit.</p></div

Mass spectrometry analysis of wild-type and mutant N-Rne proteins obtained from UV-crosslinking.

<p>(A) Partial peptide sequence of the wild-type N-Rne showing the regions of an internal standard (IS) sequence and nucleoside-bound peptides, denoted as R, P, and M sites, corresponding to an uncompetitive site (a.a. 26–37), a catalytic site (a.a. 65–71) and an allosteric site (a.a. 427–433), respectively. (B) Extracted ion chromatograms (XICs: panels a, c and e) and corrected PSM levels (panels b, d and f) of nucleoside-bound peptides and their parent peptides of wild-type N-Rne (a and b) and Q36R (c and d) and Y25A (e and f) mutants. Nucleoside-bound peptide peaks in the XICs are denoted with asterisks to the right of the symbols, R, P, and M, corresponding to the trypsin/chymotrypsin-digested peptides, ²⁶DLDIESPGHEQK³⁷, ⁶⁵HGFLPLK⁷¹, and ⁴²⁷LIEEEALK⁴³³, respectively, in the sequence of the wild-type N-Rne. The Y25A and Q36R mutants replace the R sequence with ²⁴LADLDIESPGHEQK³⁷ and ²⁶DLDIESPGHER³⁶, respectively. In the right panels, the numbers of peptide spectrum matches (PSMs) of the parental and nucleoside-bound peptides are shown in parentheses above the black and gray bars of the corrected PSM levels. The relative levels of internal standard (IS), ¹⁶VALVDGQR²³, are expressed as 100 and one unit for the calculation of relative intensity of XIC and correction of PSM levels, respectively. Tandem mass spectrometry data are given in Table S1 and Figure S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090610#pone.0090610.s001" target="_blank">File S1</a>.</p

<i>E. coli</i> strains and plasmids used in this study.

<p><i>E. coli</i> strains and plasmids used in this study.</p

Effects of mutant proteins on RNA binding activity.

<p>(A, B) Electrophoretic mobility shift assay. The 5′ end labeled p-BR13 (0.5 pmol) was incubated with increasing concentrations of purified wild-type N-Rne or Q36R or Y25A mutant protein in 20 µl of EMSA buffer, incubated on ice (A) or at room temperature (B) for 10 min, and analyzed by 12% nondenaturing PAGE. Binding constants were calculated based on slopes calculated from the graph. To avoid induction of RNA cleavage, Mg²⁺ was omitted from the EMSA reactions. (C) UV crosslinking of N-Rne-wt, N-Rne-Q36R and N-Rne-Y25A to p-BR13. Two pmol of p-BR13 was incubated with 100 pmol of N-wild-type Rne, Q36R or Y25A mutant protein in 20 µl of crosslinking buffer and exposed to UV light for 30 min. Samples were loaded onto 10% polyacrylamide gels (lanes 2, 5, 8) and samples in the absence of p-BR13 (lane 1, 4, 7) or UV irradiation (lanes 3, 6, 9) were also loaded as controls. The gel was stained with Coomassie brilliant blue and dried. The radioactivity in each band was detected using a phosphorimager and OptiQuant software. The number of crosslinked p-BR13 per pmol of protein was calculated.</p

Effects of Y25A and Q36R on the catalytic activity of RNase E <i>in vivo</i> and <i>in vitro</i>.

<p>(A) Plasmid copy number of pNRNE4, pNRNE4-Q36R and pNRNE4-Y25A in KSL2000. Plasmids were purified from KSL2000 cells harboring pNRNE4, pNRNE4-Q36R or pNRNE4-Y25A and were digested with <i>Hin</i>dIII, which has a unique cleavage site in all of the plasmids tested. Plasmid copy number was calculated relative to the concurrent presence of the pSC101 derivative (pBAD-RNE), which replicates independently of Rne, by measuring the molar ratio of the ColE1-type plasmid to the pBAD-RNE plasmid. (B) Growth characteristics of KSL2003 cells expressing wild-type N-Rne or the Q36R or Y25A mutant proteins. Growth of KSL2003 cells harboring pLAC-RNE2, pLAC-RNE2-Q36R, or pLAC-RNE2-Y25A was measured individually on LB-agar plates containing 1.0 to 1000 µM IPTG. Numbers on the top indicate the number of bacterial cells in each spot. (C) Plasmid copy number of pET28a in KSL2003. Plasmids were purified from KSL2003, KSL2003-Q36R or KSL2003-Y25A cells harboring pET28a and digested with <i>Hin</i>dIII, which has a unique cleavage site in all the plasmids tested. Plasmid copy number was calculated relative to the concurrent presence of the pSC101 derivative (pLAC-RNE2, pLAC-RNE2-Q36R or pLAC-RNE2-Y25A) by measuring the molar ratio of the ColE1-type plasmid to the pSC101-derived plasmid. (D) Expression profiles of Rne and mutant proteins in KSL2003. The membrane probed with an anti-Rne polyclonal antibody was stripped and reprobed with an anti-S1 polyclonal antibody to provide an internal standard. The relative abundance of protein was quantified by setting the amount of wild-type Rne to 1. KSL2003 cells were grown in LB medium containing 10 µM IPTG. (E) <i>In vitro</i> cleavage of p-BR13 by wild-type N-Rne, Q36R and Y25A mutant proteins. Two pmol of 5′ end-labeled p-BR13 was incubated with 1 pmol of purified wild-type N-Rne or Q36R or Y25A mutant protein in 20 µl of cleavage buffer at 37°C. Samples were removed at each indicated time point and mixed with an equal volume of loading buffer. Samples were denatured at 65°C for 5 min and loaded onto 15% polyacrylamide gel containing 8 M urea. The radioactivity in each band was quantified using a phosphorimager and OptiQuant software.</p