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T1 - Synthesis of peptide nucleic acid monomers

FIGURE 9 An outline of the chemical synthesis of nucleic acids.

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Nature Chemical BiologyVOL 6, DECEMBER 2010

Peptide nucleic acids (PNAs) are DNA analogs in which the normal phosphodiester backbone is replaced by 2-aminoethyl glycine linkages. Hybridization of PNAs with RNA or DNA follows normal rules for Watson-Crick base pairing and occurs with high affinity. Thus, PNAs are a promising choice for applications that benefit from high-affinity hybridization. They are assembled using techniques adapted from peptide chemistry. Protocols are given for both automated and manual synthesis of PNAs as well as their purification. The advantages of each method are discussed, as are the different monomers and reagents that are required. Additionally, protocols are given for adding peptides to PNAs (which can enhance hybridization or cell uptake of the PNA) and for adding a biotin label.

Nature Communications S.Y. Wu, et al., Nature Communications, 2014, 5, 3459-3459.

Eylon Yavin, Eric D. A. Stemp, Valerie L. O�Shea, Sheila S. David, and Jacqueline K. Barton
PNAS; March 7, 2006; vol. 103; no. 10; 3610-3614û

Nature Biotechnology (2013) doi:10.1038/nbt.2714

The temperature is assumed to be the critical parameter in limiting the peptide synthesis as documented in 1988 by Merrifield . A heat-induced unfolding of peptide chains can be understood as an initial point for the successful construction of large proteins, shown by the Dolphin group . Here we studied this methodology in the solid phase synthesis of peptide nucleic acids as an initial step for the entry into “personalized medicine” in oncology using exemplarily the cathepsin B gene (CtsB) and the corresponding mRNA as molecular target. The SPPS, the attachment of a fluorescence dye and the following ligation of the modules by disulfide formation result in the final CtsB-BioShuttle conjugate designed for fluorescence imaging in the selected cell lines. HeLa cells were used as a control and the imaging of the CtsB mRNA was documented . Here we confirmed the data of the non-invasive HeLa cervix carcinoma and the invasive MDA-MB-231 the breast cancer cell lines differentially expressing the CtsB (Figure ). The CtsB-BioShuttle on MDA-MB-231 resulted in strong signal using the construct with the complementary PNA I. This would be the direct way to detect tumor cells and treat with patient specific PNA at the same time. Insistently, the complexity of the pharmaceutical research and in biotechnology, the dedicated chemical methodologies like the SPPS hold a key role in the development of patient-specific drugs and imaging agents.

Ernest K. Lewis, Wade C. Haaland, Freddy Nguyen, Daniel A. Heller, Matthew J. Allen, Robert R. MacGregor, C. Scott Berger, Britain Willingham, Lori A. Burns, Graham B. I. Scott, Carter Kittrell, Bruce R. Johnson, Robert F. Curl and Michael L. Metzker
PNAS, April 12,2005, vol.102, no. 15,5346-535

Nature Biotechnology (2013) doi:10.1038/nbt.2556

24 hours before fluorescence measurement studies the cells were washed in Hank's Balanced Salts (PAN-Biotech, Germany), after treatment with trypsin/EDTA solution (0.5/0.2 %) harvested and suspended in fresh medium. Cell suspension (400 µl) were transferred to chambers of the Lab-Tek™ Chamber Slides (Nunc, USA) and incubated under identical conditions as described above. For measurement studies the MDA-MB-231 cells were treated with the CtsB-BioShuttle conjugates PNA I, PNA II, and PNA III (final concentration 100 nM) for 1 h. Finally, the medium was removed and the cells were washed with Hank's and fresh medium was added. The HeLa cells were treated identically with the CtsB-BioShuttle PNA I variant (as a negative control). After 24 hours, the samples were measured as shown in Figure .

The PNA I is the corresponding antisense molecule and possesses the antiparallel complementary sequence of the CtsB mRNA Exon I at the position 70-80. As a control we synthesized the PNA II which is identical with the sequence of the CtsB mRNA at the end of the Exon I position 70-80 . The PNA III's sequence is randomized.

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  • nucleic acids & protein synthesis notes b1 - Biology …

    Nucleic Acids Research, 2011, Vol. 39, No. 14 6277-6290 doi:10.1093/nar/gkr215

  • Nucleotide Metabolism: Nucleic Acid Synthesis

    Nature Chemical Biology Published OnLIne:15 May 2011 | DOI: 10.1038/NCHEMBIO.577

  • Nucleic Acids and Protein Synthesis All Materials ..

    Montserrat Terrazas and Eric T. Kool 346–353 Nucleic Acids Research, 2009, Vol. 37, No. 2

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Peptide synthesis: special amino acids - LifeTein

Alon A. Gorodetsky, Lars E. P. Dietrich, Paul E. Lee, Bruce Demple, Dianne K. Newman, and Jacqueline K. Barton
PNAS. 2008; 105(10): p. 3684-3689

Modifications: Stapled peptide synthesis and special amino acids

We tested the synthesized PNAs implemented into the drug delivery and targeting system called BioShuttle as functional modules for hybridization in the cells with target sequences in the cathepsin B (CtsB) mRNA reacting in case of the existence of the activated CtsB enzyme in the cells . After enzymatic cleavage in the cytoplasm the fluorescent dye Rhod110 is transported and detectable in the cell nucleus.

Applications of peptide nucleic acids - ScienceDirect

The use of PNA molecules is not restricted to its role as a DNA derivative. Derivatizations permit further functionalizations of the PNA polyamide backbone as demonstrated , . Finally the nucleobases were substituted with functional molecules as reaction partners for chemical reactions, for instance as a ligation partner in the “click-chemistry” -. Manifold ligation reactions like the copper catalyzed alkine-azide cycloadditions introduced by Sharpless are well reviewed by El-Sagheer and Brown . Here for instance, we used as ligation technology the Diels Alder Reaction with inverse electron demand (DARinv) introduced by Lindsay and comprehensively investigated by Sauer and Bertozzi , . The potential of the DARinv is well documented, undoubted and underlines multifaceted applications in the medical science. It is an attractive platform for active agents in therapy - as well as for imaging components in diagnostics , . The combination of different drugs and different diagnostic molecules in optimized ratios allows the design of molecules ready to use for bi- tri- and multi-modal strategies. These can be considered as promising molecules in the personalized medicine and in the increasing field of theranostics . Our variant, a heat-assisted approach of the solid phase peptide synthesis is considered as an indispensable methodology which realizes a proper chemistry of PNA-based backbones functionalized multifold with a great potential to contribute to the diagnostics' precision and to the better therapy's success.

Improved Synthesis Strategy for Peptide Nucleic Acids …

All this enabled the use of PNAs useful in many ways, for instance as antisense molecules in pro- as well as in eukaryotic organisms , , and as triplex forming oligonucleotides (TFO) for antigenic strategies -. From this point of view the use as therapeutic and diagnostic agents was documented . Further, PNAs have applications in analysis of biosensor chips for identification of nucleic acids . The structural and the physico-chemical properties of PNAs as well as the different synthesis methodologies are well documented -. They reveal similarities to the native nucleic acids whose phospho-ribose backbone is substituted by a backbone of poly-2-aminoaethyl glycine with nucleobases connected via an acetate linker -.

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