Conformationally preorganized peptide nucleic acids (PNAs) have been synthesized through backbone modifications at the γ-position, where R = alanine, valine, isoleucine, and phenylalanine side chains. The effects of these side-chains on the conformations and hybridization properties of PNAs were determined using a combination of CD and UV-Vis spectroscopic techniques. Our results show that the γ-position can accommodate varying degrees of sterically hindered side-chains, reaffirming the bimodal function of PNAs as the true hybrids of “peptides” and “nucleic acids.” 1. Introduction Oligonucleotides are becoming increasingly important in the postgenomic era, as molecular tools for basic research as well as potential therapeutic and diagnostic reagents for the treatment and detection of genetic diseases [1–4]. However, for many of the in vivo applications, it is not sufficed just to be able to design oligonucleotide reagents that can recognize and bind sequence specifically to DNA or RNA. These reagents would also need to be able to get into cells and withstand enzymatic degradation by nucleases in the cellular milieu. To date, diverse classes of oligonucleotide analogues have been developed, but none possesses all the characteristic features [5–8]. It is, therefore, important to be able to modify the structures and/or chemical functionalities of these reagents further, with ease and flexibility, so that many of these desired features could be augmented and/or improved upon [9, 10] and undesired attributes, such as nonspecific binding and toxicity, could be further minimized [11, 12]. A particular class of oligonucleotide analogue endowed with such synthetic flexibility is peptide nucleic acids (PNAs) [13]. PNAs are nucleic acid mimics, comprised of N-(2-aminoethyl) glycine backbone and DNA/RNA nucleobases that are connected through a flexible carboxymethylene linker. Despite the structural departure from the natural biopolymers, PNAs maintain the ability to hybridize to complementary DNA and RNA strands through Watson-Crick base-pairing, just as their natural counterparts, but with higher affinity and sequence selectivity. The improvement in binding affinity has been attributed in part to the lack of electrostatic repulsion in the backbones [14], while the enhancement in sequence selectivity has been attributed in part to the increased backbone rigidity upon hybridization as the result of solvation [15, 16]. Unlike DNA or RNA, which are prone to nucleolytic degradation, PNAs are resistant to both proteases and nucleases. These properties, together with
References
[1]
K. M. Vasquez, L. Narayanan, and P. M. Glazer, “Specific mutations induced by triplex-forming oligonucleotides in mice,” Science, vol. 290, no. 5491, pp. 530–533, 2000.
[2]
C. Potera, “Antisense—down, but not out,” Nature Biotechnology, vol. 25, no. 5, pp. 497–499, 2007.
[3]
B. A. Sullenger, “Targeted genetic repair: an emerging approach to genetic therapy,” Journal of Clinical Investigation, vol. 112, no. 3, pp. 310–311, 2003.
[4]
P. Sazani and R. Kole, “Therapeutic potential of antisense oligonucleotides as modulators of alternative splicing,” Journal of Clinical Investigation, vol. 112, no. 4, pp. 481–486, 2003.
[5]
A. De Mesmaeker, R. H？ner, P. Martin, and H. E. Moser, “Antisense oligonucleotides,” Accounts of Chemical Research, vol. 28, no. 9, pp. 366–374, 1995.
[6]
S. K. Singh, A. A. Koshkin, J. Wengel, and P. Nielsen, “LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition,” Chemical Communications, no. 4, pp. 455–456, 1998.
[7]
S. Obika, D. Nanbu, Y. Hari et al., “Stability and structural features of the duplexes containing nucleoside analogues with a fixed N-type conformation, 2'-O,4'-C-methyleneribonucleosides,” Tetrahedron Letters, vol. 39, no. 30, pp. 5401–5404, 1998.
[8]
J. E. Summerton, “Morpholinos and PNAs compared,” Letters in Peptide Science, vol. 10, no. 3-4, pp. 215–236, 2004.
[9]
R. L. Juliano and H. Yoo, “Aspects of the transport and delivery of antisense oligonucleotides,” Current Opinion in Molecular Therapeutics, vol. 2, no. 3, pp. 297–303, 2000.
[10]
I. Lebedeva, L. Benimetskaya, C. A. Stein, and M. Vilenchik, “Cellular delivery of antisense oligonucleotides,” European Journal of Pharmaceutics and Biopharmaceutics, vol. 50, no. 1, pp. 101–119, 2000.
[11]
C. A. Stein, “Keeping the biotechnology of antisense in context,” Nature Biotechnology, vol. 17, no. 3, p. 209, 1999.
[12]
B. P. Monia, “Anti-tumor activity of C-raf antisense-correction,” Nature Medicine, vol. 5, no. 2, p. 127, 1999.
[13]
P. E. Nielsen, M. Egholm, R. H. Berg, and O. Buchardt, “Sequence-selective recognition of DNA by strand displacement with thymine-substituted polyamide,” Science, vol. 254, no. 5037, pp. 1497–1500, 1991.
[14]
P. E. Nielsen, “Peptide nucleic acid. A molecule with two identities,” Accounts of Chemical Research, vol. 32, no. 7, pp. 624–630, 1999.
[15]
H. Rasmussen, S. J. Kastrup, J. N. Nielsen, J. M. Nielsen, and P. E. Nielsen, “Crystal structure of a peptide nucleic acid (PNA) duplex at 1.7 ？ resolution,” Nature Structural Biology, vol. 4, no. 2, pp. 98–101, 1997.
[16]
V. Menchise, G. De Simone, T. Tedeschi et al., “Insights into peptide nucleic acid (PNA) structural features: the crystal structure of a D-lysine-based chiral PNA-DNA duplex,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 21, pp. 12021–12026, 2003.
[17]
E. Uhlmann, A. Peyman, G. Breipohl, and D. W. Will, “PNA: synthetic polyamide nucleic acids with unusual binding properties,” Angewandte Chemie International Edition, vol. 37, no. 20, pp. 2797–2823, 1998.
[18]
F. Beck and P. E. Nielsen, Artificial DNA: Methods and Applications, vol. 2003, CRC Press, Boca Raton, Fla, USA, 2003.
[19]
V. A. Kumar and K. N. Ganesh, “Conformationally constrained PNA analogues: structural evolution toward DNA/RNA binding selectivity,” Accounts of Chemical Research, vol. 38, no. 5, pp. 404–412, 2005.
[20]
S. Pensato, M. Saviano, and A. Romanelli, “New peptide nucleic acid analogues: synthesis and applications,” Expert Opinion on Biological Therapy, vol. 7, no. 8, pp. 1219–1232, 2007.
[21]
R. Corradini, S. Sforza, T. Tedeschi, and R. Marchelli, “Chirality as a tool in nucleic acid recognition: principles and relevance in biotechnology and in medicinal chemistry,” Chirality, vol. 19, no. 4, pp. 269–294, 2007.
[22]
L. Kosynkina, W. Wang, and T. C. Liang, “A convenient synthesis of chiral peptide nucleic acid (PNA) monomers,” Tetrahedron Letters, vol. 35, no. 29, pp. 5173–5176, 1994.
[23]
Y. Wu and J. C. Xu, “Synthesis of chiral peptide nucleic acids using Fmoc chemistry,” Tetrahedron, vol. 57, no. 38, pp. 8107–8113, 2001.
[24]
T. Tedeschi, S. Sforza, R. Corradini, and R. Marchelli, “Synthesis of new chiral PNAs bearing a dipeptide-mimic monomer with two lysine-derived stereogenic centres,” Tetrahedron Letters, vol. 46, no. 48, pp. 8395–8399, 2005.
[25]
C. Dose and O. Seitz, “Convergent synthesis of peptide nucleic acids by native chemical ligation,” Organic Letters, vol. 7, no. 20, pp. 4365–4368, 2005.
[26]
E. A. Englund and D. H. Appella, “Synthesis of γ-substituted peptide nucleic acids: a new place to attach fluorophores without affecting DNA binding,” Organic Letters, vol. 7, no. 16, pp. 3465–3467, 2005.
[27]
A. Dragulescu-Andrasi, S. Rapireddy, B. M. Frezza, C. Gayathri, R. R. Gil, and D. H. Ly, “A simple γ-backbone modification preorganizes peptide nucleic acid into a helical structure,” Journal of the American Chemical Society, vol. 128, no. 31, pp. 10258–10267, 2006.
[28]
S. Rapireddy, G. He, S. Roy, B. A. Armitage, and D. H. Ly, “Strand invasion of mixed-sequence B-DNA by acridine-linked, γ-peptide nucleic acid (γ-PNA),” Journal of the American Chemical Society, vol. 129, no. 50, pp. 15596–15600, 2007.
[29]
V. Chenna, S. Rapireddy, B. Sahu, C. Ausin, E. Pedroso, and D. H. Ly, “A simple cytosine to G-clamp nucleobase substitution enables chiral γ-PNAs to invade mixed-sequence double-helical B-form DNA,” ChemBioChem, vol. 9, no. 15, pp. 2388–2391, 2008.
[30]
E. A. Englund and D. H. Appella, “γ-substituted peptide nucleic acids constructed from L-lysine are a versatile scaffold for multifunctional display,” Angewandte Chemie International Edition, vol. 46, no. 9, pp. 1414–1418, 2007.
[31]
L. Christensen, R. Fitzpatrick, B. Gildea et al., “Solid-phase synthesis of peptide nucleic acids,” Journal of Peptide Science, vol. 1, no. 3, pp. 175–183, 1995.
[32]
W. C. Johnson, In Circular Dichroism: Principles and Applications, Wiley-VCH, New York, NY, USA, 2000.
[33]
S. Sen and L. Nilsson, “MD simulations of homomorphous PNA, DNA, and RNA single strands: characterization and comparison of conformations and dynamics,” Journal of the American Chemical Society, vol. 123, no. 30, pp. 7414–7422, 2001.
[34]
P. Wittung, P. E. Nielsen, O. Buchardt, M. Egholm, and B. Norden, “DNA-like double helix formed by peptide nucleic acid,” Nature, vol. 368, no. 6471, pp. 561–563, 1994.
[35]
M. Egholm, O. Buchardt, L. Christensen et al., “PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen-bonding rules,” Nature, vol. 365, no. 6446, pp. 566–568, 1993.
[36]
M. M. Green, N. C. Peterson, T. Sato, A. Teramoto, R. Cook, and S. Lifson, “A helical polymer with a cooperative response to chiral information,” Science, vol. 268, no. 5219, pp. 1860–1866, 1995.
[37]
M. M. Green, K. S. Cheon, S. Y. Yang, J. W. Park, S. Swansburg, and W. Liu, “Chiral studies across the spectrum of polymer science,” Accounts of Chemical Research, vol. 34, no. 8, pp. 672–680, 2001.
