Rational design of novel N-alkyl-N capped biostable RNA nanostructures for efficient long-term inhibition of gene expression

Computational techniques have been used to design a novel class of RNA architecture with expected improved resistance to nuclease degradation, while showing interference RNA activity. The in silico designed structure consists of a 24–29 bp duplex RNA region linked on both ends by N-alkyl-N dimeric nucleotides (BCn dimers; n = number of carbon atoms of the alkyl chain). A series of N-alkyl-N capped dumbbell-shaped structures were efficiently synthesized by double ligation of BCn-loop hairpins. The resulting BCn-loop dumbbells displayed experimentally higher biostability than their 3′-N-alkyl-N linear version, and were active against a range of mRNA targets. We studied first the effect of the alkyl chain and stem lengths on RNAi activity in a screen involving two series of dumbbell analogues targeting Renilla and Firefly luciferase genes. The best dumbbell design (containing BC6 loops and 29 bp) was successfully used to silence GRB7 expression in HER2+ breast cancer cells for longer periods of time than natural siRNAs and known biostable dumbbells. This BC6-loop dumbbell-shaped structure displayed greater anti-proliferative activity than natural siRNAs.Instituto de Salud Carlos III [Miguel Servet Program, CP13/00211, 205024141 to M.T.]; Spanish MINECO [BIO2012–32869 and BIO2015-64802-R toM.O.]; AGAUR (toM.O.); ERCCouncil (SimDNA, grant 291433, to M.O.). M.O. is an ICREA Academia fellow. Funding for open access charge: ERC Council [grant 291433 (simDNA)].Peer ReviewedPostprint (published version

Time correlation functions of equilibrium and nonequilibrium Langevin dynamics: Derivations and numerics using random numbers

We study the time correlation functions of coupled linear Langevin dynamics without and with inertia effects, both analytically and numerically. The model equation represents the physical behavior of a harmonic oscillator in two or three dimensions in the presence of friction, noise, and an external field with both rotational and deformational components. This simple model plays pivotal roles in understanding more complicated processes. The presented analytical solution serves as a test of numerical integration schemes, its derivation is presented in a fashion that allows to be repeated directly in a classroom. While the results in the absence of fields (equilibrium) or confinement (free particle) are omnipresent in the literature, we write down, apparently for the first time, the full nonequilibrium results that may correspond, e.g., to a Hookean dumbbell embedded in a macroscopically homogeneous shear or mixed flow field. We demonstrate how the inertia results reduce to their noninertia counterparts in the nontrivial limit of vanishing mass. While the results are derived using basic integrations over Dirac delta distributions, we mention its relationship with alternative approaches involving (i) Fourier transforms, that seems advantageous only if the measured quantities also reside in Fourier space, and (ii) a Fokker--Planck equation and the moments of the probability distribution. The results, verified by numerical experiments, provide additional means of measuring the performance of numerical methods for such systems. It should be emphasized that this manuscript provides specific details regarding the derivations of the time correlation functions as well as the implementations of various numerical methods, so that it can serve as a standalone piece as part of education in the framework of stochastic differential equations and calculus.Comment: 35 pages, 5 figure

Hachimoji DNA and RNA: A genetic system with eight building blocks

Reported here are DNA and RNA-like systems built from eight (hachi-) nucleotide letters (-moji) that form four orthogonal pairs. This synthetic genetic biopolymer meets the structural requirements needed to support Darwinism, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to double the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos

Roll Resonance for a Gravity-gradient Satellite

Roll and attitude stability for gravity gradient satellite

Guidebook for analysis of tether applications

This guidebook is intended as a tool to facilitate initial analyses of proposed tether applications in space. The guiding philosophy is that a brief analysis of all the common problem areas is far more useful than a detailed study in any one area. Such analyses can minimize the waste of resources on elegant but fatally flawed concepts, and can identify the areas where more effort is needed on concepts which do survive the initial analyses. The simplified formulas, approximations, and analytical tools included should be used only for preliminary analyses. For detailed analyses, the references with each topic and in the bibliography may be useful

<i>De novo</i> synthesis of budding yeast DNA polymerase alpha and <i>POL1</i> transcription at the G₁/S boundary are not required for entrance into S phase

The POL1 gene, encoding DNA polymerase α(pol α) in Saccharomyces cerevisiae, is transiently transcribed during the cell cycle at the G1/S phase boundary. Here we show that yeast pol α is present at every stage of the cell cycle, and its level only slightly increases following the peak of POL1 transcription. POL1 mRNA synthesis driven by a GAL1 promoter can be completely abolished without affecting the growth rate of logarithmically growing yeast cultures for several cell divisions, although the amount of the pol α polypeptide drops below the physiological level. Moreover, α-factor-arrested cells can enter S phase and divide synchronously even if POL1 transcription is abolished. These results indicate that the level of yeast pol α is not rate limiting and de novo synthesis of the enzyme is not required for entrance into S phase