4 research outputs found
Hydrology needed to manage droughts: the 2015 European case
Hydrology needed to manage droughts: the 2015 European cas
Co-transcriptional Assembly of Chemically Modified RNA Nanoparticles Functionalized with siRNAs
We report a generalized methodology for the one-pot production
of chemically modified functional RNA nanoparticles during in vitro
transcription with T7 RNA polymerase. The efficiency of incorporation
of 2′-fluoro-dNTP in the transcripts by the wild type T7 RNA
polymerase dramatically increases in the presence of manganese ions,
resulting in a high-yield production of chemically modified RNA nanoparticles
functionalized with siRNAs that are resistant to nucleases from human
blood serum. Moreover, the unpurified transcription mixture can be
used for functional ex vivo pilot experiments
<i>In Silico</i> Design and Enzymatic Synthesis of Functional RNA Nanoparticles
ConspectusThe use
of RNAs as scaffolds for biomedical applications has several
advantages compared with other existing nanomaterials. These include
(i) programmability, (ii) precise control over folding and self-assembly,
(iii) natural functionalities as exemplified by ribozymes, riboswitches,
RNAi, editing, splicing, and inherent translation and transcription
control mechanisms, (iv) biocompatibility, (v) relatively low immune
response, and (vi) relatively low cost and ease of production. We
have tapped into several of these properties and functionalities to
construct RNA-based functional nanoparticles (RNA NPs). In several
cases, the structural core and the functional components of the NPs
are inherent in the same construct. This permits control over the
spatial disposition of the components, intracellular availability,
and precise stoichiometry.To enable the generation of RNA NPs,
a pipeline is being developed.
On one end, it encompasses the rational design and various computational
schemes that promote design of the RNA-based nanoconstructs, ultimately
producing a set of sequences consisting of RNA or RNA–DNA hybrids,
which can assemble into the designed construct. On the other end of
the pipeline is an experimental component, which takes the produced
sequences and uses them to initialize and characterize their proper
assembly and then test the resulting RNA NPs for their function and
delivery in cell culture and animal models. An important aspect of
this pipeline is the feedback that constantly occurs between the computational
and the experimental parts, which synergizes the refinement of both
the algorithmic methodologies and the experimental protocols. The
utility of this approach is depicted by the several examples described
in this Account (nanocubes, nanorings, and RNA–DNA hybrids).
Of particular interest, from the computational viewpoint, is that
in most cases, first a three-dimensional representation of the assembly
is produced, and only then are algorithms applied to generate the
sequences that will assemble into the designated three-dimensional
construct. This is opposite to the usual practice of predicting RNA
structures from a given sequence, that is, the RNA folding problem.
To be considered is the generation of sequences that upon assembly
have the proper intra- or interstrand interactions (or both). Of particular
interest from the experimental point of view is the determination
and characterization of the proper thermodynamic, kinetic, functionality,
and delivery protocols. Assembly of RNA NPs from individual single-stranded
RNAs can be accomplished by one-pot techniques under the proper thermal
and buffer conditions or, potentially more interestingly, by the use
of various RNA polymerases that can promote the formation of RNA NPs
cotransciptionally from specifically designed DNA templates.Also of importance is the delivery of the RNA NPs to the cells
of interest <i>in vitro</i> or <i>in vivo</i>.
Nonmodified RNAs rapidly degrade in blood serum and have difficulties
crossing biological membranes due to their negative charge. These
problems can be overcome by using, for example, polycationic lipid-based
carriers. Our work involves the use of bolaamphiphiles, which are
amphipathic compounds with positively charged hydrophilic head groups
at each end connected by a hydrophobic chain. We have correlated results
from molecular dynamics computations with various experiments to understand
the characteristics of such delivery agents
Triggering of RNA Interference with RNA–RNA, RNA–DNA, and DNA–RNA Nanoparticles
Control over cellular delivery of different functionalities and their synchronized activation is a challenging task. We report several RNA and RNA/DNA-based nanoparticles designed to conditionally activate the RNA interference in various human cells. These nanoparticles allow precise control over their formulation, stability in blood serum, and activation of multiple functionalities. Importantly, interferon and pro-inflammatory cytokine activation assays indicate the significantly lower responses for DNA nanoparticles compared to the RNA counterparts, suggesting greater potential of these molecules for therapeutic use