Synthesis of Oligodeoxynucleotides with 5′‐Caps Binding RNA Targets
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- Literature Cited
Abstract
Protocols for the synthesis of oligodeoxynucleotides with a short peptidyl substituent linked to the 5??O ?terminus through a phosphodiester bond are presented. The example given is a peptidyl cap consisting of the residues of L ?prolinol, glycine, and the acyl residue of oxolinic acid. DNA probes with this cap, also known as ogOA cap, give melting point increases for duplexes with RNA targets and improve mismatch discrimination at the terminus. The cap is either introduced in one step, using a newly developed phosphoramidite reagent, or assembled on the DNA chain. The step?wise assembly of the peptidyl chain is advantageous for combinatorial studies aimed at the optimization of a cap structure. The block coupling method, introducing the preassembled cap in one step, is attractive for routine use of a cap already optimized for a given application. Cap?bearing probes can increase fidelity of hybridization in a genomic context. They can be synthesized by automated DNA synthesis. Curr. Protoc. Nucleic Acid Chem. 51:4.53.1?4.53.21. © 2012 by John Wiley & Sons, Inc.
Keywords: oligonucleotides; phosphoramidite; peptides; controlled pore glass; hybridization probes
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Basic Protocol 1: Synthesis of a Molecular Cap Building Block
- Alternate Protocol 1: Synthesis of 2′‐Oligodeoxyribonucleotides with 5′‐Cap Build Up on Solid Support
- Support Protocol 1: Synthesis of N‐Fmoc‐L‐Prolinol Linker Phosphoramidite
- Commentary
- Literature Cited
- Figures
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Basic Protocol 1: Synthesis of a Molecular Cap Building Block
Materials
Basic Protocol 2:
Materials
Alternate Protocol 1: Synthesis of 2′‐Oligodeoxyribonucleotides with 5′‐Cap Build Up on Solid Support
Materials
Support Protocol 1: Synthesis of N‐Fmoc‐L‐Prolinol Linker Phosphoramidite
Materials
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Figure 4.53.1 Enhancing selectivity and fidelity of base pairing at the terminus through a molecular cap. The cap binds to the terminal base pair, bridging the duplex, when the correct Watson‐Crick base pair is formed. The increase in duplex stability is most probably a consequence of bridging that holds together the two strands and reduces fraying at the terminus of the duplex. View Image -
Figure 4.53.2 Structures of duplexes with 5′‐caps. Sequences and structural details of the cap residues are shown in the upper part of each graphic; three‐dimensional structure are shown in the lower part of each graphic. Structural images were generated with VMD, using coordinates from PDB database entries 1ON5 (cholic acid cap), 1C95 (tryptophan cap), 1KSE (oxolinic acid cap), and 1X6W (trimethoxystilbene cap). The DNA is shown in gray and the stacking moiety of the caps are shown as orange line drawings with smaller line width. View Image -
Figure 4.53.3 Structures of self‐complementary DNA duplexes with 3′‐caps. Sequences and structural details of the cap residues are shown in the upper part of each image, and three‐dimensional structures are shown in the lower part of each graphic. Structures of duplexes were generated with VMD, using data from the PDB database entries 2BQ2 and 2KK5. The DNA is shown in gray and the stacking moiety of the caps are shown as orange line drawings with smaller line width. View Image -
Figure 4.53.4 Synthesis of the phosphoramidite building block for the ogOA cap (S.1 ). View Image -
Figure 4.53.5 Synthesis of cap‐bearing oligonucleotide S.5 via coupling of S.1 to preassembled octamer S.6 as a representative example of the final stages of a synthesis on solid support. The coupling may either be performed on a DNA synthesizer or manually, as described below. View Image -
Figure 4.53.6 Synthesis of cap‐bearing oligonucleotide via peptide coupling steps on solid support. After coupling of a linker phosphoramidite (S.8 ) to preassembled octamer S.6 , the cap is elaborated step‐wise. While a specific example is show here, a variety of different amino acid and carboxylic acid building blocks may be used to assemble different cap structures. The synthesis of linker phosphoramidite S.8 is described in the . For the synthesis of support S.6 , please see Figure . View Image -
Figure 4.53.7 Analytical data for 5′‐capped oligonucleotide S.5 . Left‐hand side: RP‐HPLC chromatogram (for conditions, see ); right‐hand side: MALDI‐TOF mass spectrum. View Image
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