Henrik M. Pfundheller

A novel class of conformationally restricted oligonucleotide analogues: synthesis of 2',3'-bridged monomers and RNA-selective hybridisation

A novel 2′,3′-bicyclic nucleoside 5 has been synthesised and incorporated into oligonucleotide analogues resulting in strong and selective binding to an RNA complement.

Synthesis of 2′-O,3′-C-linked bicyclic nucleosides and bicyclic oligonucleotides

The 3′-C-allyl furanose 4 has been used as a precursor for synthesis of the novel 2′-O,3′-C-linked bicyclic thymine nucleosides 15, 16, 20 and 25. The three bicyclic β-nucleosides 15, 20 and 25 have been incorporated into oligodeoxynucleotides. One of these nucleosides, dioxabicyclo[3.3.0]octane derivative 25, induces increased thermal stability of duplexes towards complementary RNA.

The ProbeLibrary™ - Expression profiling 99% of all human genes using only 90 dual-labeled real-time PCR Probes

While quantitative real-time RT-PCR is in principle a simple technique, the assay design remains fairly complex and designed assays often perform inadequately with respect to specificity, sensitivity, and reproducibility (1,2). The time spent on assay design, optimization, and validation often becomes a bottleneck in the implementation of new assays for large-scale expression profiling. Commercially available pre-validated real-time RT-PCR assays simplify the assay development process, but the time of delivery sometimes causes delays in experimental progress. Furthermore, prevalidated probe-based assays lack flexibility, due to the fact that these assays target a specific site in a given transcript. Consequently, quantification of another transcript or splice variant requires a different assay. Here we describe a novel, highly flexible concept for quantitative real-time RT-PCR, based on the development of a ProbeLibraryTM of 90 prevalidated real-time PCR detection probes, and a new web-based assay design software, enabling fast and easy design of optimal real-time PCR assays for gene expression analysis. By combining individual ProbeLibraryTM probes and target-specific PCR primers selected using the Primer3 software (3), the Assay Design Center software is able to design more than 644,000 different assays in the human transcriptome or target 98% of all human transcripts (Figure 1, Table 1). PRINCIPLES OF THE TECHNOLOGY

Oligonucleotides Containing Novel 4′‐C‐ or 3′‐C‐(Aminoalkyl)‐Branched Thymidines

The synthesis of four novel 3′-C-branched and 4′-C-branched nucleosides and their transformation into the corresponding 3′-O-phosphoramidite building blocks for automated oligonucleotide synthesis is reported. The 4′-C-branched key intermediate 11 was synthesized by a convergent strategy and converted to its 2′-O-methyl and 2′-deoxy-2′-fluoro derivatives, leading to the preparation of novel oligonucleotide analogues containing 4′-C-(aminomethyl)-2′-O-methyl monomer X and 4′-C-(aminomethyl)-2′-deoxy-2′-fluoro monomer Y (Schemes 2 and 3). In general, increased binding affinity towards complementary single-stranded DNA and RNA was obtained with these analogues compared to the unmodified references (Table 1). The presence of monomer X or monomer Y in a 2′-O-methyl-RNA oligonucleotide had a negative effect on the binding affinity of the 2′-O-methyl-RNA oligonucleotide towards DNA and RNA. Starting from the 3′-C-allyl derivative 28, 3′-C-(3-aminopropyl)-protected nucleosides and 3′-O-phosphoramidite derivatives were synthesized, leading to novel oligonucleotide analogues containing 3′-C-(3-aminopropyl)thymidine monomer Z or the corresponding 3′-C-(3-aminopropyl)-2′-O,5-dimethyluridine monomer W (Schemes 4 and 5). Incorporation of the 2′-deoxy monomer Z induced no significant changes in the binding affinity towards DNA but decreased binding affinity towards RNA, while the 2′-O-methyl monomer Z induced decreased binding affinity towards DNA as well as RNA complements (Table 2).

Evaluation of Oligonucleotides Containing Two Novel 2′-O-Methyl Modified Nucleotide Monomers: A 3′-C-Allyl and a 2′-O-3′-C-Linked Bicyclic Derivative

The two ribo-configured nucleosides 1-(3-C-allyl-2-O-methyl-beta-D-ribo-pentofuranosyl)thymine 3 and (1S,5R,6R,8R)-5-hydroxy-6-(hydroxymethyl)-1-methoxy-8-(thymin-1-yl )- 2,7-dioxabicyclo[3.3.0]octane 6 have been transformed into their corresponding phosphoramidites, 5 and 8 respectively, and used as building blocks for the synthesis of modified oligonucleotides. The oligonucleotides were shown to hybridize with decreased binding affinity towards complementary single stranded DNA and RNA.

Locked Nucleic Acids: Synthesis and Characterization of LNA‐T Diol

Locked nucleic acids (LNAs) are RNA derivatives that have an O-methylene linkage between the 2 and 4 positions of the ribose. This leads to exceptionally high-affinity binding to complementary sequences. LNAs are synthesized from a commercially available sugar, 1,2:5,6-di-O-isopropylidene-a-D-allofuranose. An efficient and simplified procedure is presented for synthesizing a glycol donor that can be used for synthesis of a variety of LNA monomers. Then, as an example, the synthesis of the thymidine analog of LNA from this glycol donor is presented. The protocols give high yields of the desired products and avoid the use of time-consuming column chromatography.

Synthesis of novel 3'-C-branched 2'-deoxynucleosides. Incorporation of 3'-C-(3-hydroxypropyl)thymidine into oligodeoxynucleotides

The methyl glycoside derivatives 4, 6, 10 and 32 have been used as precursors for the synthesis of novel 3′-C-alkyl-modified α- and β-2′-deoxynucleosides. Using an alternative linear strategy, 3′-C-methyl- and 3′-C-azidomethyl-modified thymidines 16 and 17 have been synthesized. Hybridization experiments with oligodeoxynucleotides containing 3′-C-(3-hydroxypropyl)thymidine monomers are reported.

Synthesis and hybridization properties of β- and α-oligodeoxynucleotides containing β- and α-1-(3-C-allyl-2-deoxy-D-erythro-pentofuranosyl)thymine and α-1-[3-C-(3-aminopropyl)-2-deoxy-D-erythro-pentofuranosyl]thymine

Convergent synthesis of β- and α-1-(3-C-allyl-2-deoxy-D-erythro-pentofuranosyl)thymine and their incorporation into β- and α-oligodeoxynucleotides (ODNs) is described. The thermal stabilities of duplexes formed between modified ODNs and complementary single-stranded DNA and RNA have been evaluated. In all cases stable duplexes are formed, but whereas β-ODNs containing β-3′-C-allylthymidine show moderately lowered thermal stability towards both DNA and RNA, α-ODNs containing α-3′-C-allylthymidine show significantly increased thermal stabilities compared with the corresponding β-ODN reference duplexes. Even more stable duplexes towards both DNA and RNA have been obtained using an α-ODN containing one α-1-[3-C-(3-aminopropyl)-2-deoxy-D-erythro-pentofuranosyl]thymine monomer.