6 research outputs found
Carbohydrate-Functionalized Locked Nucleic Acids: Oligonucleotides with Extraordinary Binding Affinity, Target Specificity, and Enzymatic Stability
Three different C5-carbohydrate-functionalized
LNA uridine phosphoramidites
were synthesized and incorporated into oligodeoxyribonucleotides.
C5-Carbohydrate-functionalized LNA display higher affinity toward
complementary DNA/RNA targets (Δ<i>T</i><sub>m</sub>/modification up to +11.0 °C), more efficient discrimination
of mismatched targets, and superior resistance against 3′-exonucleases
compared to conventional LNA. These properties render C5-carbohydrate-functionalized
LNAs as promising modifications in antisense technology and other
nucleic acid targeting applications
Recognition of Mixed-Sequence DNA Duplexes: Design Guidelines for Invaders Based on 2′‑<i>O</i>‑(Pyren-1-yl)methyl-RNA Monomers
The development of
agents that recognize mixed-sequence double-stranded
DNA (dsDNA) is desirable because of their potential as tools for detection,
regulation, and modification of genes. Despite progress with triplex-forming
oligonucleotides, peptide nucleic acids, polyamides, and other approaches,
recognition of mixed-sequence dsDNA targets remains challenging. Our
laboratory studies <i>Invaders</i> as an alternative approach
toward this end. These double-stranded oligonucleotide probes are
activated for recognition of mixed-sequence dsDNA through modification
with +1 interstrand zippers of intercalator-functionalized nucleotides
such as 2′-<i>O</i>-(pyren-1-yl)methyl-RNA monomers
and have recently been shown to recognize linear dsDNA, DNA hairpins,
and chromosomal DNA. In the present work, we systematically studied
the influence that the nucleobase moieties of the 2′-<i>O</i>-(pyren-1-yl)methyl-RNA monomers have on the recognition
efficiency of Invader duplexes. Results from thermal denaturation,
binding energy, and recognition experiments using Invader duplexes
with different +1 interstrand zippers of the four canonical 2′-<i>O</i>-(pyren-1-yl)methyl-RNA <b><u>A</u></b>/<b><u>C</u></b>/<b><u>G</u></b>/<b><u>U</u></b> monomers show that
incorporation of these motifs is a general strategy for activation
of probes for recognition of dsDNA. Probe duplexes with interstrand
zippers comprising <b><u>C</u></b> and/or <b><u>U</u></b> monomers result in the most efficient
recognition of dsDNA. The insight gained from this study will drive
the design of efficient Invaders for applications in molecular biology,
nucleic acid diagnostics, and biotechnology
C5-Alkynyl-Functionalized α‑L‑LNA: Synthesis, Thermal Denaturation Experiments and Enzymatic Stability
Major efforts are currently being
devoted to improving the binding
affinity, target specificity, and enzymatic stability of oligonucleotides
used for nucleic acid targeting applications in molecular biology,
biotechnology, and medicinal chemistry. One of the most popular strategies
toward this end has been to introduce additional modifications to
the sugar ring of affinity-inducing conformationally restricted nucleotide
building blocks such as locked nucleic acid (LNA). In the preceding
article in this issue, we introduced a different strategy toward this
end, i.e., C5-functionalization of LNA uridines. In the present article,
we extend this strategy to α-L-LNA: i.e., one of the most interesting
diastereomers of LNA. α-L-LNA uridine monomers that are conjugated
to small C5-alkynyl substituents induce significant improvements in
target affinity, binding specificity, and enzymatic stability relative
to conventional α-L-LNA. The results from the back-to-back articles
therefore suggest that C5-functionalization of pyrimidines is a general
and synthetically straightforward approach to modulate biophysical
properties of oligonucleotides modified with LNA or other conformationally
restricted monomers
Synthesis and Biophysical Properties of C5-Functionalized LNA (Locked Nucleic Acid)
Oligonucleotides modified with conformationally
restricted nucleotides
such as locked nucleic acid (LNA) monomers are used extensively in
molecular biology and medicinal chemistry to modulate gene expression
at the RNA level. Major efforts have been devoted to the design of
LNA derivatives that induce even higher binding affinity and specificity,
greater enzymatic stability, and more desirable pharmacokinetic profiles.
Most of this work has focused on modifications of LNA’s oxymethylene
bridge. Here, we describe an alternative approach for modulation of
the properties of LNA: i.e., through functionalization of LNA nucleobases.
Twelve structurally diverse C5-functionalized LNA uridine (U) phosphoramidites
were synthesized and incorporated into oligodeoxyribonucleotides (ONs),
which were then characterized with respect to thermal denaturation,
enzymatic stability, and fluorescence properties. ONs modified with
monomers that are conjugated to small alkynes display significantly
improved target affinity, binding specificity, and protection against
3′-exonucleases relative to regular LNA. In contrast, ONs modified
with monomers that are conjugated to bulky hydrophobic alkynes display
lower target affinity yet much greater 3′-exonuclease resistance.
ONs modified with C5-fluorophore-functionalized LNA-U monomers enable
fluorescent discrimination of targets with single nucleotide polymorphisms
(SNPs). In concert, these properties render C5-functionalized LNA
as a promising class of building blocks for RNA-targeting applications
and nucleic acid diagnostics
Identification and Characterization of Second-Generation Invader Locked Nucleic Acids (LNAs) for Mixed-Sequence Recognition of Double-Stranded DNA
The
development of synthetic agents that recognize double-stranded
DNA (dsDNA) is a long-standing goal that is inspired by the promise
for tools that detect, regulate, and modify genes. Progress has been
made with triplex-forming oligonucleotides, peptide nucleic acids,
and polyamides, but substantial efforts are currently devoted to the
development of alternative strategies that overcome the limitations
observed with the classic approaches. In 2005, we introduced Invader
locked nucleic acids (LNAs), i.e., double-stranded probes that are
activated for mixed-sequence recognition of dsDNA through modification
with “+1 interstrand zippers” of 2′-<i>N</i>-(pyren-1-yl)methyl-2′-amino-α-l-LNA monomers.
Despite promising preliminary results, progress has been slow because
of the synthetic complexity of the building blocks. Here we describe
a study that led to the identification of two simpler classes of Invader
monomers. We compare the thermal denaturation characteristics of double-stranded
probes featuring different interstrand zippers of pyrene-functionalized
monomers based on 2′-amino-α-l-LNA, 2′-<i>N</i>-methyl-2′-amino-DNA, and RNA scaffolds. Insights
from fluorescence spectroscopy, molecular modeling, and NMR spectroscopy
are used to elucidate the structural factors that govern probe activation.
We demonstrate that probes with +1 zippers of 2′-<i>O</i>-(pyren-1-yl)methyl-RNA or 2′-<i>N</i>-methyl-2′-<i>N</i>-(pyren-1-yl)methyl-2′-amino-DNA monomers recognize
DNA hairpins with similar efficiency as original Invader LNAs. Access
to synthetically simple monomers will accelerate the use of Invader-mediated
dsDNA recognition for applications in molecular biology and nucleic
acid diagnostics
4′‑<i>C</i>‑Methoxy-2′-deoxy-2′-fluoro Modified Ribonucleotides Improve Metabolic Stability and Elicit Efficient RNAi-Mediated Gene Silencing
We designed novel 4′-modified
2′-deoxy-2′-fluorouridine (2′-F U) analogues
with the aim to improve nuclease resistance and potency of therapeutic
siRNAs by introducing 4′-<i>C</i>-methoxy (4′-OMe)
as the alpha (C4′α) or beta (C4′β) epimers.
The C4′α epimer was synthesized by a stereoselective
route in six steps; however, both α and β epimers could
be obtained by a nonstereoselective approach starting from 2′-F
U. <sup>1</sup>H NMR analysis and computational investigation of the
α-epimer revealed that the 4′-OMe imparts a conformational
bias toward the <i>North</i>-<i>East</i> sugar
pucker, due to intramolecular hydrogen bonding and hyperconjugation
effects. The α-epimer generally conceded similar thermal stability
as unmodified nucleotides, whereas the β-epimer led to significant
destabilization. Both 4′-OMe epimers conferred increased nuclease
resistance, which can be explained by the close proximity between
4′-OMe substituent and the vicinal 5′- and 3′-phosphate
group, as seen in the X-ray crystal structure of modified RNA. siRNAs
containing several C4′α-epimer monomers in the sense
or antisense strands triggered RNAi-mediated gene silencing with efficiencies
comparable to that of 2′-F U