Transcript Synthesis and Characterization of Peptide Nucleic Acid for

Synthesis and Characterization of Peptide Nucleic Acid for Experimental Use of Directing Anthrapyrazole Derivatives.
James M. Bradley, Dr. Frank Guziec, and Dr. Kerry Bruns
Department of Chemistry and Biochemistry, Southwestern University, Georgetown, TX 78626
Abstract
Purification
Peptide nucleic acids (PNAs) are uncharged and achiral DNA analogues known
to exhibit enhanced binding and specificity for single-stranded and doublestranded nucleic acids. These synthetic pseudo-peptides lack the sugarphosphate backbone of traditional nucleic acids and can be designed to
compliment specific sequences in DNA. Our study aims to couple the sequence
specific binding properties of PNAs with the intercalating capabilities of
anthrapyrazole (AP) derivatives to see if we can direct the intercalator to a
specific region of DNA. As a result, these AP-PNA adducts may display qualities
favorable for prolonged gene-silencing or gene-activation. Our PNA was
designed to compliment the T7 promoter sequence of PBluescriptKII and thereby
bind to the DNA in vitro. The synthesis of the PNA was carried out using 9fluorenylmethoxycarbonyl solid phase peptide synthesis (FmocSPPS), and the
structure of the PNA was verified using mass spectrometry with a theoretical
molecular weight of 3471.0g/mol and an experimental result of 3478.5g/mol.
With its identification confirmed, the compound was purified and collected using a
high performance liquid chromatography (HPLC) preparative reversed phase
column. After purification, a multi-ether spacer was added to the PNA with
FmocSPPS, and a biotin-labeled lysine residue was added to about half of the
product for further analysis of the intercalator-free compound. A dot blot
immunoassay using streptavidin-(horseradish peroxidase) conjugates was
conducted on the biotinylated PNA in order to find observable concentrations of
he compound for further analysis. In future experiments we plan to see how
addition of an intercalator changes binding characteristics by comparing the APPNA adducts with the AP-free PNA in an electrophoretic gel shift assay. If the
intercalator significantly enhances the PNA’s binding ability, then further research
with AP-PNA adducts in vivo may reveal this compound’s potential as a gene
therapeutic.
Intercalator-free and biotinylated PNA was isolated using a Beckman
System Gold 126NM Solvent Module, System Gold 166NM Detector,
and SC100 Fraction Collector. The identity of the PNA was confirmed
via mass spectrometry by BioSynthesis, Inc. in Louisville, TX.
Dot Blot Analysis
E. Coli cells containing the pBluescriptKII plasmid with the T7 promoter
sequence were grown, and the DNA was harvested and quantitated
using a Shimadzu UV-1601 UV-Visible Spectrophotometer according
to the procedure described by Sambrook and Russell (6), as well as
digested by EcoR1, Pvu2, and Bssh2. The DNA was cross-linked to a
neutral nylon membrane via UV radiation, and denatured in basic
conditions
Interpretation
Figure 1.
Scheme 1.
Comparison of the backbones of DNA
and PNA. B = nucleobases adenine,
cytosine, guanine, and thymine.
Reprinted from ref 7. Copyright 1996
Verlagsgesellschaft mbH.
General cycle FmocSPPS and
cleavage of finished product.
Reprinted from ref 8. Copyright
1991 Rainin Instrument
Company, Inc.
Introduction
In the age of the genome, many diseases and disorders can be attributed to the
over-expression of specific genes, even some forms of cancer (1);
countermeasures to moderate these disorders include the control of gene
expression with DNA-binding small molecules or anti-sense, RNA interfering,
small molecules. Even more, small molecules such as these may even be
effective anti-viral therapeutics by neutralizing the RNA genome of devastating
retro viruses (2). One class of such small molecules is PNAs. These differ from
traditional nucleic acids’ structure by having a backbone consisting of N-(2aminoethyl)glycine units, see figure1. The lack of negatively charged phosphate
groups in the backbone rid this compound of electrostatic repulsive forces, as
well as avoid recognition from nucleases. This enables PNAs to have high
binding abilities with complimentary sequences and possibly inhibit biological
effects (3).
Given that acridine-PNA adducts increase dsDNA binding efficiency without the
expense of target specificity, will AP-PNA adducts exhibit similar characteristics
(4).
Experimental Approach
Synthesis of PNA
FmocSPPS was carried out using a Labortec AG Peptide Synthesizer SP4000.
Fmoc and Bhoc protected monomers from Applied Biosystems and Fluka were
used according to the procedure described by Chan and White (5).
Discussion
With the identity of our PNA confirmed, it is necessary to determine if it
does bind to the T7 promoter sequence. Adding the biotin moiety to
the PNA will enable facile detection for future experiments by utilizing
streptavidin-(horseradish peroxidase) conjugates. Research is still
underway to see if the biotinylated PNA will hybridize with the
denatured T7 promoter sequence. Once the binding characteristics of
our intercalator-free PNA is well defined, we shall then synthesize APPNA adducts using FmocSPPS and determine the intercalators effect
on binding efficiency and speficity.
References
With the advent of FmocSPPS, designing custom peptides is not only feasible but
cost-efficient. Following scheme 1, a peptide can be produced from monomers
and chemically bonded in a specific order to attain the desired compound.
Purpose
The peak at 3478.5g/mol in the mass spectrometry corresponds to our
desired PNA; the peak at 5734.1g/mol is the insulin used as a
reference by BioSynthesis, see figure 2.
Figure 2.
Mass to charge ratio versus percent transmittance.
Peaks at 3478.5g/mol and 5734.1g/mol.
(1) Okamoto, Y.; Ozaki, T.; Miyazaki, K.; Aoyama, M.; Miyazaki, M.; Nakagawara, A.
UbcH10 is the cancer-related E2 ubiquitin-conjugating enzyme. Cancer Res. 2003, 63,
4167-4173.
(2) Nulf, C. J.; Corey, D. Intracellular inhibition of hepatitis C virus (HCV) internal
ribosomal entry site (IRES)-dependent translation by peptide nucleic acids (PNAs)
and locked nucleic acids (LNAs). Nucleic Acids Res. 2004, 32, 3792-3798.
(3) Christensen, L.; Fitzpatrick, R.; Gildea, B.; Petersen, K. H.; Hansen, H. F.; Koch, T.;
Egholm, M.; Buchardt, O.; Nielsen, P. E.; Coull, J.; Berg, R. H. Solid-phase
synthesis of peptide nucleic acids. Journal of Peptide Science 1995, 3, 175-183.
(4) Nielsen, P.; Bentin, T. Superior duplex DNA strand invasion by acridine conjugated
peptide nucleic acids. JACS Communications 2003, 125, 6378-6379.
(5) Chan, W.; White, P., eds. Fmoc Solid Phase Peptide Synthesis: A Practical Approach;
Oxford University Press: New York, 2000.
(6) Sambrook, J.; Russell, D. Molecular Cloning: A Laboratory Manual; 3rd ed.; Cold
Spring Harbor Laboratory Press: New York, 2001.
(7) Haaima, G.; Lohse, A.; Buchardt, O.; Nielsen, P. Peptide nucleic acids (PNAs)
containing thymine monomers eerived from chiral amino acids: Hybridization and
solubility properties of D-lysine PNA. Angew. Chem. Int. Ed. Engl. 1996, 35, 19391942.
(8) Protein Technologies Model PS3 Automated Peptide Synthesizer; Rainin Instrument
Company, Inc.: Woburn, MA, 1991.
Acknowledgements
We would like to thank the Welch Foundation for funding our research
with grant number AF-0005. We thank Dr. Martín Gonzalez and Dr.
Maha Zewail-Foote for their time and generosity, and we are grateful
for the facilities at BioSynthesis. I would also like to thank Dr. Kerry
Bruns and Dr. Frank Guziec for their tutorage and for this research
opportunity.