Tertiary structure of supercoiled DNA is a significant factor in a number of genetic functions and is apparently affected by environmental conditions. We applied atomic force microscopy (AFM) for imaging the supercoiled DNA deposited at different ionic conditions. We have employed a technique for the sample preparation that permits high-resolution AFM imaging of DNA bound to the surface in buffer solutions without drying the sample (AFM in situ). The AFM data show that at low ionic strength, DNA molecules are loosely interwound supercoils with an irregular shape. Plectonemic superhelices are formed in high-concentration, nearphysiological salt solutions. At such ionic conditions, superhelical loops are typically separated by regions of close helixhelix contacts. The data obtained show directly and unambiguously that overall geometry of supercoiled DNA depends dramatically on ionic conditions. This fact and the formation of close contacts between DNA helices are important features of supercoiled DNA related to its biological functions.DNA supercoiling is thought to play key roles in genetic processes in the cell (1). One fundamental feature of DNA supercoiling is that distantly separated DNA regions can be brought in close proximity. Bringing two distant sites into proximity is required for DNA recombination (2-5), for the control of DNA supercoiling by DNA topoisomerases (6-8), and for gene regulation through DNA looping (9-11). A conventional model for supercoiled DNA is a plectonemic helix in which both strands interwind each other. However, DNA is a highly charged polymer, so that electrostatic repulsion of negatively charged DNA helices opposes folding and especially formation of close contacts between DNA regions. Counterions shield the negative charge of DNA and hence decrease the repulsion between DNA segments. So the following questions arise. Does the geometry of supercoiled DNA depend on ionic strength? If it does, then to what extent do salt conditions change the geometry of supercoiled DNA? Finally, what is the geometry of supercoiled DNA at different ionic conditions? These questions are of great importance because the configurations of supercoiled DNA are ultimately related to its functions.Monte Carlo simulations of supercoiling (12-18) have shown that ion concentration has a strong effect on conformation of supercoiled DNA. In particular, systematic theoretical studies of the effect of ionic strength on the geometry of supercoiled DNA were performed by Vologodskii and Cozzarelli (15,16). Experimental data on the salt-dependent geometry of supercoiled DNA are quite controversial. Electron microscopy (EM) was successfully applied to studies of DNA supercoiling, and systematic studies of the structure of supercoiled DNA were performed by Boles et al. (19). They found no changes in geometry of molecules when spreading conditions were changed from a low salt buffer to a buffer containing 10 mM MgCl 2 , which is equivalent to a high salt solution (20). On the contrary, light scattering (21, 22)...

Summary One of the advantages of nanotechnology is the feasibility to construct therapeutic particles carrying multiple therapeutics with defined structure and stoichiometry. The field of RNA nanotechnology is emerging. However, controlled assembly of stable RNA nanoparticles with multiple functionalities which retain their original role is challenging due to refolding after fusion. Herein, we report the construction of thermodynamically stable X-shaped RNA nanoparticles to carry four therapeutic RNA motifs by self-assembly of reengineered small RNA fragments. We proved that each arm of the four helices in the X-motif can harbor one siRNA, ribozyme, or aptamer without affecting the folding of the central pRNA-X core, and each daughter RNA molecule within the nanoparticle folds into their respective authentic structures and retains their biological and structural function independently. Gene silencing effects were progressively enhanced as the number of the siRNA in each pRNA-X nanoparticles gradually increased from one to two, three, and four. More importantly, systemic injection of ligand-containing nanoparticles into the tail-vein of mice revealed that the RNA nanoparticles remained intact and strongly bound to cancers without entering the liver, lung or any other organs or tissues, while remaining in cancer tissue for more than 8 h.

This work reports the design of polymer micelles with cross-linked ionic cores that display high stability. Block ionomer complexes were utilized as a micellar template for the synthesis of the cross-linked micelles. Such micelles represent hydrophilic nanospheres of core-shell morphology. The core comprises a network of the cross-linked polyanions, which is surrounded by the shell of hydrophilic PEO chains.

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The HIV-1 restriction factor SAMHD1 is a tetrameric enzyme activated by guanine nucleotides with dNTP triphosphate hydrolase activity (dNTPase). In addition to this established activity, there have been a series of conflicting reports as to whether the enzyme also possesses single-stranded DNA and/or RNA 3′-5′ exonuclease activity. SAMHD1 was purified using three chromatography steps, over which the DNase activity was largely separated from the dNTPase activity, but the RNase activity persisted. Surprisingly, we found that catalytic and nucleotide activator site mutants of SAMHD1 with no dNTPase activity retained the exonuclease activities. Thus, the exonuclease activity cannot be associated with any known dNTP binding site. Monomeric SAMHD1 was found to bind preferentially to single-stranded RNA, while the tetrameric form required for dNTPase action bound weakly. ssRNA binding, but not ssDNA, induces higher-order oligomeric states that are distinct from the tetrameric form that binds dNTPs. We conclude that the trace exonuclease activities detected in SAMHD1 preparations arise from persistent contaminants that co-purify with SAMHD1 and not from the HD active site. An in vivo model is suggested where SAMHD1 alternates between the mutually exclusive functions of ssRNA binding and dNTP hydrolysis depending on dNTP pool levels and the presence of viral ssRNA.