9 research outputs found
C2′-Pyrene-Functionalized Triazole-Linked DNA: Universal DNA/RNA Hybridization Probes
Development of universal hybridization probes, that is,
oligonucleotides
displaying identical affinity toward matched and mismatched DNA/RNA
targets, has been a longstanding goal due to potential applications
as degenerate PCR primers and microarray probes. The classic approach
toward this end has been the use of “universal bases”
that either are based on hydrogen-bonding purine derivatives or aromatic
base analogues without hydrogen-bonding capabilities. However, development
of probes that result in truly universal hybridization without compromising
duplex thermostability has proven challenging. Here we have used the
“click reaction” to synthesize four C2′-pyrene-functionalized
triazole-linked 2′-deoxyuridine phosphoramidites. We demonstrate
that oligodeoxyribonucleotides modified with the corresponding monomers
display (a) minimally decreased thermal affinity toward DNA/RNA complements
relative to reference strands, (b) highly robust universal hybridization
characteristics (average differences in thermal denaturation temperatures
of matched vs mismatched duplexes involving monomer <b>W</b> are <1.7 °C), and (c) exceptional affinity toward DNA targets
containing abasic sites opposite of the modification site (Δ<i>T</i><sub>m</sub> up to +25 °C). The latter observation,
along with results from absorption and fluorescence spectroscopy,
suggests that the pyrene moiety is intercalating into the duplex whereby
the opposing nucleotide is pushed into an extrahelical position. These
properties render C2′-pyrene-functionalized triazole-linked
DNA as promising universal hybridization probes for applications in
nucleic acid chemistry and biotechnology
Synthesis, Hybridization Characteristics, and Fluorescence Properties of Oligonucleotides Modified with Nucleobase-Functionalized Locked Nucleic Acid Adenosine and Cytidine Monomers
Conformationally
restricted nucleotides such as locked nucleic
acid (LNA) are very popular as affinity-, specificity-, and stability-enhancing
modifications in oligonucleotide chemistry to produce probes for nucleic
acid targeting applications in molecular biology, biotechnology, and
medicinal chemistry. Considerable efforts have been devoted in recent
years to optimize the biophysical properties of LNA through additional
modification of the sugar skeleton. We recently introduced C5-functionalization
of LNA uridines as an alternative and synthetically more straightforward
approach to improve the biophysical properties of LNA. In the present
work, we set out to test the generality of this concept by studying
the characteristics of oligonucleotides modified with four different
C5-functionalized LNA cytidine and C8-functionalized LNA adenosine
monomers. The results strongly suggest that C5-functionalization of
LNA pyrimidines is indeed a viable approach for improving the binding
affinity, target specificity, and/or enzymatic stability of LNA-modified
ONs, whereas C8-functionalization of LNA adenosines is detrimental
to binding affinity and specificity. These insights will impact the
future design of conformationally restricted nucleotides for nucleic
acid targeting applications
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 Double-Stranded DNA Using Energetically Activated Duplexes Modified with N2′-Pyrene‑, Perylene‑, or Coronene-Functionalized 2′‑<i>N</i>‑Methyl-2′-amino-DNA Monomers
Invader probes have been proposed
as alternatives to polyamides,
triplex-forming oligonucleotides, and peptide nucleic acids for recognition
of chromosomal DNA targets. These double-stranded probes are activated
for DNA recognition by +1 interstrand zippers of pyrene-functionalized
nucleotides. This particular motif forces the intercalating pyrene
moieties into the same region, resulting in perturbation and destabilization
of the probe duplex. In contrast, the two probe strands display very
high affinity toward complementary DNA. The energy difference between
the probe duplexes and recognition complexes provides the driving
force for DNA recognition. In the present study, we explore the properties
of Invader probes based on larger intercalators, i.e., perylene and
coronene, expecting that the larger π-surface area will result
in additional destabilization of the probe duplex and further stabilization
of probe–target duplexes, in effect increasing the thermodynamic
driving force for DNA recognition. Toward this end, we developed protocols
for 2′-<i>N</i>-methyl-2′-amino-2′-deoxyuridine
phosphoramidites that are functionalized at the N2′-position
with pyrene, perylene, or coronene moieties and incorporated these
monomers into oligodeoxyribonucleotides (ONs). The resulting ONs and
Invader probes are characterized by thermal denaturation experiments,
analysis of thermodynamic parameters, absorption and fluorescence
spectroscopy, and DNA recognition experiments. Invader probes based
on large intercalators efficiently recognize model targets
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
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