Proposed mechanism of “non-ladder type” by-product formation.

<p>Proposed mechanism of “non-ladder type” by-product formation.</p

Agarose gel analysis of the PCR products of the 8 SELEX cycles (lanes 1–8) showing the ladder-type and non-ladder-type by-products.

<p>Visualization by ethidium bromide staining; 4% agarose gel.</p

Cloning and sequencing of the library from selection cycle 8 yielded different classes of oligonucleotide sequences.

<p>Cloning and sequencing of the library from selection cycle 8 yielded different classes of oligonucleotide sequences.</p

Proposed mechanism of “ladder type” by-product formation.

<p>Proposed mechanism of “ladder type” by-product formation.</p

PCR amplification of representative mono-clones, the start library and a non-template control (NTC).

<p>Visualization by ethidium bromide staining; 4% agarose gel.</p

Filter retention assay, A), with radioactive labelled DNA from the start library, 7^th and 11^th selection cycle.

<p>A strong increase in binding affinity is observable, demonstrating a successful enrichment during the SELEX. <b>B</b>) Sequencing data from the 11^th selection cycle without primer binding sites. All mono-clones share the previously for high affinity APC aptamers described consensus motive (5′-TATCMCGNATGGGS-3′) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114693#pone.0114693-Mayer1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114693#pone.0114693-Tolle1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114693#pone.0114693-Mller1" target="_blank">[19]</a>.</p

Large Scale Synthesis of 2′-Amino-LNA Thymine and 5‑Methylcytosine Nucleosides

Thymine intermediate <b>17</b> has been synthesized on a multigram scale (50 g, 70 mmol) from starting sugar <b>1</b> in 15 steps in an overall yield of 73%, with only 5 purification steps. The key thymine intermediate <b>18</b> was obtained from <b>17</b> in a single step in 96% yield, whereas the key 5-methylcytosine intermediate <b>20</b> was obtained from <b>17</b> in 2 steps in 58% yield. This highly efficient large scale route necessitates only 2 and 3 novel steps to obtain N2′-functionalized thymine and 5-methylcytosine amino-LNA phosphoramidites from these key intermediates, respectively

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

FISH detection of <i>H.pylori</i> in paraffin-embedded sections of gastric biopsies, using 2’-O-methyl/LNA FISH detection conditions.

<p>(A) Detection of <i>H. pylori</i> using the HP_LNA/2OMe_PS probe in a histological slide of a gastric biopsy specimen of an infected patient; (B) Experiment control performed in parallel using the HP_LNA/2OMe_PS probe in a histological slide of a gastric biopsy specimen of an non infected patient. Arrows indicate the presence of <i>H. pylori</i> infecting the gastric mucosa. All images were taken at equal exposure times Scale bars: 10 µm. Original magnification of ×1000.</p

FISH detection of <i>H. pylori</i> 26695 strain (ATCC 700392) using FAM- HP_LNA/2OMe_PO and HP_LNA/2OMe_PS probes.

<p>FISH analysis was performed by epifluorescent microscopy in smears, using either 50% (vol/vol) formamide and 4 M urea as denaturing agents in the hybridization buffer. Smears without probe were used as negative control (Control) (a). (2B) Average fluorescence intensity from each probe in 4 M urea and 50% formamide (v/v) buffers; fluorescent signal intensity is expressed in arbitrary fluorescent units (AFU) and was quantified using the by ImageJ software. All images were acquired at equal exposure conditions. Original magnification: 1000x.</p