3 research outputs found
Automatic Pseudorotaxane Formation Targeting on Nucleic Acids Using a Pair of Reactive Oligodeoxynucleotides
Here we report a
novel method to form a pseudorotaxane architecture
using only a pair of reactive oligodeoxyribonucleotides (ODNs), which
we designed and synthesized, and then performed the pseudorotaxane
formation reaction with both DNA and RNA oligonucleotides. The reaction
proceeded smoothly without any extra reagents at 37 °C and pH
7.2, leading to the formation of a stable complex on a denaturing
polyacrylamide gel. Interestingly, the pseudorotaxane was formed with
the cyclized ODN reversibly by the slipping process. This new pseudorotaxane
formation represents a promising method for developing new DNA nanotechnologies
and antisense oligonucleotides
Structure-Guided Control of siRNA Off-Target Effects
Short interfering
RNAs (siRNAs) are promising therapeutics that
make use of the RNA interference (RNAi) pathway, but liabilities arising
from the native RNA structure necessitate chemical modification for
drug development. Advances in the structural characterization of components
of the human RNAi pathway have enabled structure-guided optimization
of siRNA properties. Here we report the 2.3 Ă
resolution crystal
structure of human Argonaute 2 (hAgo2), a key nuclease in the RNAi
pathway, bound to an siRNA guide strand bearing an unnatural triazolyl
nucleotide at position 1 (g1). Unlike natural nucleotides, this analogue
inserts deeply into hAgo2âs central RNA binding cleft and thus
is able to modulate pairing between guide and target RNAs. The affinity
of the hAgo2âsiRNA complex for a seed-only matched target was
significantly reduced by the triazolyl modification, while the affinity
for a fully matched target was unchanged. In addition, siRNA potency
for off-target repression was reduced (4-fold increase in IC<sub>50</sub>) by the modification, while on-target knockdown was improved (2-fold
reduction in IC<sub>50</sub>). Controlling siRNA on-target versus
microRNA (miRNA)-like off-target potency by projection of substituent
groups into the hAgo2 central cleft from g1 is a new approach to enhance
siRNA selectivity with a strong structural rationale
Short Interfering RNA Guide Strand Modifiers from Computational Screening
Short interfering RNAs (siRNAs) are
promising drug candidates for
a wide range of targets including those previously considered âundruggableâ.
However, properties associated with the native RNA structure limit
drug development, and chemical modifications are necessary. Here we
describe the structure-guided discovery of functional modifications
for the guide strand 5â˛-end using computational screening with
the high-resolution structure of human Ago2, the key nuclease on the
RNA interference pathway. Our results indicate the guide strand 5â˛-end
nucleotide need not engage in WatsonâCrick (W/C) H-bonding
but must fit the general shape of the 5â˛-end binding site in
MID/PIWI domains of hAgo2 for efficient knockdown. 1,2,3-Triazol-4-yl
bases formed from the CuAAC reaction of azides and 1-ethynylribose,
which is readily incorporated into RNA via the phosphoramidite, perform
well at the guide strand 5â˛-end. In contrast, purine derivatives
with modified Hoogsteen faces or N2 substituents are poor choices
for 5â˛-end modifications. Finally, we identified a 1,2,3-triazol-4-yl
base incapable of W/C H-bonding that performs well at guide strand
position 12, where base pairing to target was expected to be important.
This work expands the repertoire of functional nucleotide analogues
for siRNAs