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>_m 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>_m/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>_m/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