4 research outputs found
Synthesis and Characterization of Oligodeoxyribonucleotides Modified with 2′-Amino-α‑l‑LNA Adenine Monomers: High-Affinity Targeting of Single-Stranded DNA
The
development of conformationally restricted nucleotide building
blocks continues to attract considerable interest because of their
successful use within antisense, antigene, and other gene-targeting
strategies. Locked nucleic acid (LNA) and its diastereomer α-l-LNA are two interesting examples thereof. Oligonucleotides
modified with these units display greatly increased affinity toward
nucleic acid targets, improved binding specificity, and enhanced enzymatic
stability relative to unmodified strands. Here we present the synthesis
and biophysical characterization of oligodeoxyribonucleotides (ONs)
modified with 2′-amino-α-l-LNA adenine monomers <b>W</b>–<b>Z</b>. The synthesis of the target phosphoramidites <b>1</b>–<b>4</b> is initiated from pentafuranose <b>5</b>, which upon Vorbrüggen glycosylation, O2′-deacylation,
O2′-activation and C2′-azide introduction yields nucleoside <b>8</b>. A one-pot tandem Staudinger/intramolecular nucleophilic
substitution converts <b>8</b> into 2′-amino-α-l-LNA adenine intermediate <b>9</b>, which after a series
of nontrivial protecting-group manipulations affords key intermediate <b>15</b>. Subsequent chemoselective N2′-functionalization
and O3′-phosphitylation give targets <b>1</b>–<b>4</b> in ∼1–3% overall yield over 11 steps from <b>5</b>. ONs modified with pyrene-functionalized 2′-amino-α-l-LNA adenine monomers <b>X</b>–<b>Z</b> display
greatly increased affinity toward DNA targets (Δ<i>T</i><sub>m</sub>/modification up to +14 °C). Results from absorption
and fluorescence spectroscopy suggest that the duplex stabilization
is a result of pyrene intercalation. These characteristics render
N2′-pyrene-functionalized 2′-amino-α-l-LNAs of considerable interest for DNA-targeting applications
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