Background: Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication including shuttle RNA, mainly mRNA and microRNA. As exosomes naturally carry RNA between cells, these particles might be useful in gene cancer therapy to deliver therapeutic short interfering RNA (siRNA) to the target cells. Despite the promise of RNA interference (RNAi) for use in therapy, several technical obstacles must be overcome. Exogenous siRNA is prone to degradation, has a limited ability to cross cell membranes and may induce an immune response. Naturally occurring RNA carriers, such as exosomes, might provide an untapped source of effective delivery strategies. Results: This study demonstrates that exosomes can deliver siRNA to recipient cells in vitro. The different strategies were used to introduce siRNAs into human exosomes of various origins. The delivery of fluorescently labeled siRNA via exosomes to cells was confirmed using confocal microscopy and flow cytometry. Two different siRNAs against RAD51 and RAD52 were used to transfect into the exosomes for therapeutic delivery into target cells. The exosome-delivered siRNAs were effective at causing post-transcriptional gene silencing in recipient cells. Moreover, the exosome-delivered siRNA against RAD51 was functional and caused the massive reproductive cell death of recipient cancer cells. Conclusions: The results strongly suggest that exosomes effectively delivered the siRNA into the target cells. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated in vitro by the strong knockdown of RAD51, a prospective therapeutic target for cancer cells. The results give an additional evidence of the ability to use human exosomes as vectors in cancer therapy, including RNAi-based gene therapy.

This communication reports an analysis of Tn 5 /IS 50 target site selection by using an extensive collection of Tn 5 and IS 50 insertions in two relatively small regions of DNA (less than 1 kb each). For both regions data were collected resulting from in vitro and in vivo transposition events. Since the data sets are consistent and transposase was the only protein present in vitro , this demonstrates that target selection is a property of only transposase. There appear to be two factors governing target selection. A target consensus sequence, which presumably reflects the target selection of individual pairs of Tn 5 /IS 50 bound transposase protomers, was deduced by analyzing all insertion sites. The consensus Tn 5 /IS 50 target site is A-GNTYWRANC-T. However, we observed that independent insertion sites tend to form groups of closely located insertions (clusters), and insertions very often were spaced in a 5-bp periodic fashion. This suggests that Tn 5 /IS 50 target selection is facilitated by more than two transposase protomers binding to the DNA, and, thus, for a site to be a good target, the overlapping neighboring DNA should be a good target, too. Synthetic target sequences were designed and used to test and confirm this model.

Transposition is a multistep process in which a transposable element DNA sequence moves from its original genetic location to a new site. Early steps in this process include the formation of a transposition complex in which the end sequences of the transposable element are brought together in a structurally precise fashion through the action of the element-encoded transposase protein and the cleavage of the element free from the adjoining DNA. If transposition complex formation must precede DNA cleavage (or nicking), then changing the length of the donor DNA between closely spaced ends should have dramatic effects on the frequency of the transposition. This question has been examined by studying the effects of altering donor DNA length on IS50 transposition. Donor DNA < or = 64 bp severely impaired transposition. Donor DNA > or = 200 bp demonstrated high transposition frequencies with only modest length dependencies. Constructs with donor DNA lengths between 66 and 174 bp demonstrated a dramatic periodic effect on transposition (periodicity approximately 10.5 bp).

The radA gene is an archaeal homolog of bacterial recA and eukaryotic RAD51 genes, which are critical components in homologous recombination and recombinational DNA repair. We cloned the radA gene from a hyperthermophilic archaeon, Pyrobaculum islandicum, overproduced the radA gene product in Escherichia coli and purified it to homogeneity. The purified P. islandicum RadA protein maintained its secondary structure and activities in vitro at high temperatures, up to 87 °C. It also showed high stability of 18.3 kcal·mol⁻¹ (76.5 kJ·mol⁻¹) at 25 °C and neutral pH. P. islandicum RadA exhibited activities typical of the family of RecA‐like proteins, such as the ability to bind ssDNA, to hydrolyze ATP in a DNA‐dependent manner and to catalyze DNA strand exchange. At 75 °C, all DNAs tested stimulated ATPase activity of the RadA. The protein exhibited a break in the Arrhenius plot of ATP hydrolysis at 75 °C. The cooperativity of ATP hydrolysis and ssDNA‐binding ability of the protein above 75 °C were higher than at lower temperatures, and the activation energy of ATP hydrolysis was lower above this break point temperature. These results suggest that the ssDNA‐dependent ATPase activity of P. islandicum RadA displays a temperature‐dependent capacity to exist in two different catalytic modes, with 75 °C being the critical threshold temperature.