Oligonucleotide-based therapy has become an alternative to classical approaches in the search of novel therapeutics involving gene-related diseases. Several mechanisms have been described in which demonstrate the pivotal role of oligonucleotide for modulating gene expression. Antisense oligonucleotides (ASOs) and more recently siRNAs and miRNAs have made important contributions either in reducing aberrant protein levels by sequence-specific targeting messenger RNAs (mRNAs) or restoring the anomalous levels of non-coding RNAs (ncRNAs) that are involved in a good number of diseases including cancer. In addition to formulation approaches which have contributed to accelerate the presence of ASOs, siRNAs and miRNAs in clinical trials; the covalent linkage between non-viral vectors and nucleic acids has also added value and opened new perspectives to the development of promising nucleic acid-based therapeutics. This review article is mainly focused on the strategies carried out for covalently modifying siRNA and miRNA molecules. Examples involving cell-penetrating peptides (CPPs), carbohydrates, polymers, lipids and aptamers are discussed for the synthesis of siRNA conjugates whereas in the case of miRNA-based drugs, this review article makes special emphasis in using antagomiRs, locked nucleic acids (LNAs), peptide nucleic acids (PNAs) as well as nanoparticles. The biomedical applications of siRNA and miRNA conjugates are also discussed.

Fluoropyrimidines, such as 5-fluorouracil (5-FU) and related prodrugs, are considered one of the most successful agents in the treatment of colorectal cancer, yet poor specificity and tumor cell resistance remain the major limiting bottlenecks. Here, we exploited for the first time the ability of two DNA nanoscaffolds, a DNA tetrahedron (Td) and rectangle DNA origami, to incorporate 5-fluoro-2'-deoxyuridine (FdUn) oligomers. In addition, cholesterol moieties were synthetically attached to Td and DNA origami staples to enhance cellular uptake. DNA nanostructures functionalized with FdUn exhibited an enhanced cytotoxicity and higher ability to trigger apoptosis in colorectal cancer cells relative to conventional 5-FU and FdU, especially having cholesterol as an internalization helper. The cholesterol content mostly correlates with the increase of the FdUn nanostructure cytotoxicity. DNA nanoscaffolds bearing FdUn were able to circumvent the low sensitivity of colorectal cancer cells towards 5-FU. Both DNA nanostructures attained a comparable cytotoxic effect yet Td displays higher antiproliferative action. The ability to reduce the proliferation of cancer cells is mainly related to the concentration of DNA nanostructures. The present work suggests that self-assembled DNA nanoparticles are privileged vehicles for delivering fluoropyrimidines, opening new avenues to the development of promising therapeutics for cancer treatment.

Compaction of negatively charged polyanions by polycations with different characteristics is investigated using Monte Carlo simulation in a coarse-grain model. Two different routes are tested and the results compared. In one, the polycation/polyanion charge ratio is varied by increasing the amount of polycations, keeping all the chain characteristics constant. In the other, the linear charge density of the polycations is altered but their number is kept constant. The set of systems in which the linear charge density changes is used as a model for a system comprising chains with different degrees of ionization under different pH conditions. In both cases, polycation/polyanion charge ratios ranging from 0.25 to 1.25 are addressed. The system with unitary charge ratio is common to both routes. It is seen that, although the overall trends followed by the two sets of systems are similar, marked differences can be discerned both for low charge ratios, and for the higher ones, where the systems are overcharged. Coexistence regimes are clearly detected in some of the systems. The results obtained computationally can be used to guide practical applications.

This work addresses the impact of pH variation on DNA-polyethylenimine (PEI) complex formation, in aqueous solution and at constant ionic strength. An initial potentiometric characterization of the acid-base behavior of PEI is carried out to measure the concentration of ionized species in the relevant systems. The characterization of the DNA-PEI complexes is performed by precipitation assays, agarose gel electrophoresis, photon correlation spectroscopy, and zeta potential analysis. It is observed that the variations on the electrophoretic mobility, size, and electrical properties of complexes display nonmonotonic, nontrivial trends with pH, if the same polycation/polyanion charge ratios are used for different values of pH. It is seen that both linear charge density and the relative number of chains of the condensing agent are important factors governing the condensation behavior. Complexes prepared at pH 4, for example, indicate strong binding and a large mean size, while those prepared at pH 8 are smaller, in a more uniform population. Finally, charge inversion was observed for all studied pH values (even below charge neutralization).

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Efficient DNA condensation and decondensation, as well as low toxicity, are required for an efficient gene delivery vehicle. We report on the condensation of DNA by a mixture of cationic agents, low-molecular-weight polyethylenimine (PEI, 1.2 KDa) and Fe(III) ions, and respective decondensation, using experimental and theoretical methods. It was found that a significant reduction in the amount of PEI necessary to induce DNA condensation is achieved by the addition of the trivalent ions, which are very inefficient on their own. In addition, the mixture makes DNA decompaction by heparin easier, starting from similar degrees of condensation. The results obtained using simulations of coarse-grain models are coherent with those obtained experimentally. It was also found that the improved effect of the multivalent ions is related to the preferred positioning of the trivalent ions in the DNA areas less populated by the polycation chains, in between the polycation chains and at the ends of the DNA, which facilitates the overall condensation.

A direct connection between the PAZ/3′-overhang binding affinity and the siRNA potency and specificity is defined through complementary experimental and computational results.

Centaurea solstitialis L. (yellow starthistle, Asteraceae) is a Eurasian native plant introduced as an exotic into North and South America, and Australia, where it is regarded as a noxious invasive. Changes in ploidy level have been found to be responsible for numerous plant biological invasions, as they are involved in trait shifts critical to invasive success, like increased growth rate and biomass, longer life-span, or polycarpy. C. solstitialis had been reported to be diploid (2n = 2x = 16 chromosomes), however, actual data are scarce and sometimes contradictory. We determined for the first time the absolute nuclear DNA content by flow cytometry and estimated ploidy level in 52 natural populations of C. solstitialis across its native and non-native ranges, around the world. All the C. solstitialis populations screened were found to be homogeneously diploid (average 2C value of 1.72 pg, SD = ±0.06 pg), with no significant variation in DNA content between invasive and non-invasive genotypes. We did not find any meaningful difference among the extensive number of native and non-native C. solstitialis populations sampled around the globe, indicating that the species invasive success is not due to changes in genome size or ploidy level.