The Structure of DNA within Cationic Lipid/DNA Complexes

The structure of DNA within CLDCs used for gene delivery is controversial. Previous studies using CD have been interpreted to indicate that the DNA is converted from normal B to C form in complexes. This investigation reexamines this interpretation using CD of model complexes, FTIR as well as Raman spectroscopy and molecular dynamics simulations to address this issue. CD spectra of supercoiled plasmid DNA undergo a significant loss of rotational strength in the signal near 275 nm upon interaction with either the cationic lipid dimethyldioctadecylammonium bromide or 1,2-dioleoyltrimethylammonium propane. This loss of rotational strength is shown, however, by both FTIR and Raman spectroscopy to occur within the parameters of the B-type conformation. Contributions of absorption flattening and differential scattering to the CD spectra of complexes are unable to account for the observed spectra. Model studies of the CD of complexes prepared from synthetic oligonucleotides of varying length suggest that significant reductions in rotational strength can occur within short stretches of DNA. Furthermore, some alteration in the hydrogen bonding of bases within CLDCs is indicated in the FTIR and Raman spectroscopy results. In addition, alterations in base stacking interactions as well as hydrogen bonding are suggested by molecular dynamics simulations. A global interpretation of all of the data suggests the DNA component of CLDCs remains in a variant B form in which base/base interactions are perturbed

A Stopped-Flow Kinetic Study of the Assembly of Nonviral Gene Delivery Complexes

Stopped-flow circular dichroism and fluorescence spectroscopy are used to characterize the assembly of complexes consisting of plasmid DNA bound to the cationic lipids dimethyldioctadecylammonium bromide and 1, 2-dioleoyl- 3-trimethylammonium-propane and a series of polyamidoamine dendrimers. The kinetics of complexation determined from the stopped-flow circular dichroism measurements suggests complexation occurs within 50 ms. Further analysis, however, was precluded by the presence of mixing (shear) artifacts. Stopped-flow fluorescence employing the high-affinity DNA dyes Hoechst 33258 and YOYO-1 was able to resolve two sequential steps in the assembly of complexes that are assigned to binding/dehydration and condensation events. The rates of each process were determined over the temperature range of 10–50°C and activation energies were determined from the slope of Arrhenius plots. The behavior of polyamidoamine dendrimers can be separated into two classes based on their differing binding modes: generation 2 and the larger generations (G4, G7, and G9). The larger generations have activation energies for binding that follow the trend G4 > G7 > G9. The activation energies for condensation (compaction) of complexes composed of these same dendrimers have the opposite trend G9 > G7 > G4. It is postulated that a balance between a more energetically favorable condensation and less favorable binding may prove beneficial in enhancing gene delivery

The Structure of DNA within Cationic Lipid/DNA Complexes

The structure of DNA within CLDCs used for gene delivery is controversial. Previous studies using CD have been interpreted to indicate that the DNA is converted from normal B to C form in complexes. This investigation reexamines this interpretation using CD of model complexes, FTIR as well as Raman spectroscopy and molecular dynamics simulations to address this issue. CD spectra of supercoiled plasmid DNA undergo a significant loss of rotational strength in the signal near 275 nm upon interaction with either the cationic lipid dimethyldioctadecylammonium bromide or 1,2-dioleoyltrimethylammonium propane. This loss of rotational strength is shown, however, by both FTIR and Raman spectroscopy to occur within the parameters of the B-type conformation. Contributions of absorption flattening and differential scattering to the CD spectra of complexes are unable to account for the observed spectra. Model studies of the CD of complexes prepared from synthetic oligonucleotides of varying length suggest that significant reductions in rotational strength can occur within short stretches of DNA. Furthermore, some alteration in the hydrogen bonding of bases within CLDCs is indicated in the FTIR and Raman spectroscopy results. In addition, alterations in base stacking interactions as well as hydrogen bonding are suggested by molecular dynamics simulations. A global interpretation of all of the data suggests the DNA component of CLDCs remains in a variant B form in which base/base interactions are perturbed

Search for singly and pair-produced leptoquarks coupling to third-generation fermions in proton-proton collisions at root s=13 TeV

A search for leptoquarks produced singly and in pairs in proton-proton collisions is presented. We consider the leptoquark (LQ) to be a scalar particle of charge -1/3e coupling to a top quark plus a tau lepton (t tau) or a bottom quark plus a neutrino (b nu), or a vector particle of charge +2/3e, coupling to t nu or b tau. These choices are motivated by models that can explain a series of anomalies observed in the measurement of B meson decays. In this analysis the signatures t tau nu b and t tau nu are probed, using data recorded by the CMS experiment at the CERN LHC at root s = 13 TeV and that correspond to an integrated luminosity of 137 fb(-1). These signatures have not been previously explored in a dedicated search. The data are found to be in agreement with the standard model prediction. Lower limits at 95% confidence level are set on the LQ mass in the range 0.98-1.73 TeV, depending on the LQ spin and its coupling lambda to a lepton and a quark, and assuming equal couplings for the two LQ decay modes considered. These are the most stringent constraints to date on the existence of leptoquarks in this scenario. (C) 2021 The Author(s). Published by Elsevier B.V.Peer reviewe