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 citrate-capped AuNP (1.25-fold) and AuNP-DNA with a nonspecific poly(T) sequence (1.25-fold). We showed that both nonspecific 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.

DNA-conjugated gold nanoparticles (AuNPs) have been shown to enhance the translation of mRNA. However, the specific sequence on the DNA dictates the specific mRNA to be enhanced. This study describes poly(thymine)-functionalized AuNPs (AuNP-p(T)DNA) capable of enhancing the translation of any mRNA template that is incorporated into pcDNA6 vector with bovine growth hormone (BGH) polyadenylation signal (P(A)). We demonstrated this by incorporating four genes: green fluorescence protein (GFP), general control nonderepressible 5 (GCN5), cAMP-responsive element binding protein 1 (CREB1), and X-box-binding protein 1-spliced (XBP-1S) separately into pcDNA6 vector with BGH P(A) before their expression in HeLa lysate. The addition of AuNP-p(T)DNA to HeLa lysate containing GFP, GCN5, CREB1, and XBP-1S mRNA increased protein synthesis 1.80, 1.99, 1.95, and 2.20 times, respectively. Similar translation enhancement was also observed in a multiplex reaction containing the mRNA of three genes together in the lysate. Complementary p(T)DNA hybridization to the poly(A) tail of the mRNA was critical as the removal of either p(T)DNA or BGH P(A) in XBP-1S mRNA or the replacement of p(T)DNA with p(A)DNA reduced the translation back to baseline level. Finally, an optimum length of 25 nucleotides for the DNA oligomer and a AuNP-p(T)DNA:mRNA ratio of 0.658 achieved a 3.08-fold translation enhancement. The AuNP-p(T)DNA nanoconstruct could be incorporated into commercial cell-free protein synthesis kits as a universal translation enhancer.

Therapeutic peptides suffer from major drawbacks such as peptide degradation in vivo due to proteolysis. Gold nanoparticles (AuNPs) are an effective carrier for therapeutic peptides that improve their stability in vivo, while also enabling nonspecific adsorption of complementary proteins to enhance their effectiveness. Using p53 peptide as a model known to disrupt the intracellular MDM2-p53 protein–protein interaction which tags the endogenous p53 proteins for degradation, we conjugated p53 peptides to AuNPs (AuNP-p53) and examined the functionality of AuNP-p53 to release the endogenous p53 proteins from being tagged for degradation, thereby increasing the level of stable p53 proteins in acute myeloid leukemia 2 (AML2) cells. We found that AuNPs did not just protect conjugated p53 peptides from trypsin degradation, but also helped to recruit 56.5% and 26.4% of total MDM2 and p53 proteins in the cells to form a protein corona around AuNP-p53. The proximity of MDM2/p53 complexes and p53 peptide on the surface of AuNP-p53 facilitated the action of p53 peptides to cause a sustained elevation of the p53 level in AML2 cells up to 6 h, which was not possible with free p53 peptide alone at the same concentration. Even a 20-fold higher concentration of free p53 peptide caused only a short-lived elevated p53 level of 1 h. The outcome of this study highlights the utility of combining conjugated ligands and complementary protein adsorption on nanoparticles to improve the biological functionality of the therapeutic ligands.

Absrmct-In this paper we report the results of our study on the behaviour and implementation of the Parent Centric Operator (PCX) within the Generalized Generation Gap (G3) model using five test functions of 10, 20 and 50 dimensions. Our study indicates that G3-PCX performs fairly well on most functions, hut its performance is not good for highly nonlinear, multidimensional problems (Rastrigin, Ackley, Griewangk). We observed the same behaviour of G3-PCX while designing a 22 element Yagi-Uda Antenna for gain maximization (known to he a highly nonlinear problem). We derived a simple variant G3-PCX-I1 using a Roulette wheel based parent selection scheme which performs better than G3-PCX on the highly nonlinear multidimensional problems.

We have previously shown the use of gold nanoparticles (AuNPs) functionalized with DNA (AuNP-DNA) to increase insulin mRNA translation in a cell-free system. In this study, we translate the concept into a whole cell system to demonstrate functionality despite the additional complexity of intracellular delivery and mRNA translation inside living cells. We selected an insulin-secreting pancreatic islet cell line, RIN-5F, as our model and designed a DNA oligomer (insDNA) that is complementary to the 3′-untranslated region of insulin mRNA for conjugation to AuNPs (AuNP-insDNA). AuNP-insDNA was stable in the extracellular environment of RIN-5F cells for up to 24 h, without eliciting any cell toxicity. Upon cellular entry, AuNP-insDNA was able to sustain enhanced insulin secretion from 6 to 12 h post-incubation, peaking at 10 h with an enhancement factor of 1.69-fold. This enhancement was not observed when insDNA was removed or replaced with poly thymine or poly adenine DNAs. The enhanced insulin secreted was 100% functional and capable of binding to its insulin receptor. The outcome of this study demonstrated the feasibility of AuNP-DNA to enhance the synthesis of proteins in whole cells and could serve as a new direction of invoking a patient’s own beta cells to increase insulin secretion for treatment of diabetes.