Synthetic molecules that recognize specific sequences within cellular DNA are potentially powerful tools for investigating chromosome structure and function. Here, we designed antigene peptide nucleic acids (agPNAs) to target the transcriptional start sites for the human progesterone receptor B (hPR-B) and A (hPR-A) isoforms at sequences predicted to be single-stranded within the open complex of chromosomal DNA. We found that the agPNAs were potent inhibitors of transcription, showing for the first time that synthetic molecules can recognize transcription start sites inside cells. Breast cancer cells treated with agPNAs showed marked changes in morphology and an unexpected relationship between the strictly regulated levels of hPR-B and hPR-A. We confirmed these phenotypes using siRNAs and antisense PNAs, demonstrating the power of combining antigene and antisense strategies for gene silencing. agPNAs provide a general approach for controlling transcription initiation and a distinct option for target validation and therapeutic development.

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The recognition of cellular nucleic acids by synthetic oligonucleotides is a versatile strategy for regulating biological processes. The vast majority of published studies have focused on antisense oligonucleotides that target mRNA, but it is also possible to design antigene oligonucleotides that are complementary to chromosomal DNA. Antigene oligomers could be used to inhibit the expression of any gene or analyze promoter structure and the mechanisms governing gene regulation. Other potential applications of antigene oligomers include activation of expression of chosen genes or the introduction of mutations to correct genetic disease. Peptide nucleic acid (PNA) is a nonionic DNA/RNA mimic that possesses outstanding potential for recognition of duplex DNA. Here we describe properties of PNAs and the challenges for their development as robust antigene agents.

Epigallocatechin-3-O-gallate (EGCG) is the major catechin component of green tea (Cameria sinensis), and is known to possess antiviral activities against a wide range of DNA viruses and RNA viruses. However, few studies have examined chemical modifications of EGCG in terms of enhanced antiviral efficacy. This paper discusses which steps of virus infection EGCG interferes with, citing previous reports. EGCG appears most likely to inhibits the early stage of infections, such as attachment, entry, and membrane fusion, by interfering with viral membrane proteins. According to the relationships between structure and antiviral activity of catechin derivatives, the 3-galloyl and 5′-OH group of catechin derivatives appear critical to antiviral activities. Enhancing the binding affinity of EGCG to virus particles would thus be important to increase virucidal activity. We propose a newly developed EGCG-fatty acid derivative in which the fatty acid on the phenolic hydroxyl group would be expected to increase viral and cellular membrane permeability. EGCG-fatty acid monoesters showed improved antiviral activities against different types of viruses, probably due to their increased affinity for virus and cellular membranes. Our study promotes the application of EGCG-fatty acid derivatives for the prevention and treatment of viral infections.

Peptide nucleic acids (PNAs) are a powerful tool for recognition of double-stranded DNA. Strand invasion is most efficient when pyrimidine PNAs are linked to form a bisPNA in which one strand binds by Watson-Crick base pairing while the other binds by Hoogsteen base pairing to the newly formed PNA-DNA duplex. Within many genes, however, polypyrimidine target sequences may not be located in optimal positions relative to transcription factor binding sites, and this deficiency may complicate attempts to identify potent antigene PNAs. To increase the versatility of strand invasion by PNAs, we have synthesized bisPNAs and bisPNA-peptide conjugates containing a mixed base extension of the Watson-Crick polypyrimidine strand. We find that these tail-clamp PNAs (TC-PNAs) bind duplex DNA and inhibit transcription. DNA recognition occurs with single-stranded or TC-bisPNAs and requires attachment of positively charged amino acids. Association rate constants, k(a), for binding to DNA by TC-PNAs are as high as 35000 M(-1) s(-1) and are usually only a fewfold lower than for analogous PNAs that lack mixed base extensions. The ability to bind duplex DNA is not always necessary for inhibition of transcription, possibly because PNAs can bind to accessible DNA within the transcription bubble created by RNA polymerase. These results, together with similar findings independently obtained by Nielsen and colleagues [Bentin, T., Larsen, H. J., and Nielsen, P. E. (2003) Biochemistry 42, 13987-13995], expand the range of sequences within duplex DNA that are accessible to PNAs and suggest that TC-PNA-peptide conjugates are good candidates for further testing as antigene agents.

Peptide nucleic acids (PNAs) offer a distinct option for silencing gene expression in mammalian cells. However, the full value of PNAs has not been realized, and the rules governing the recognition of cellular targets by PNAs remain obscure. Here we examine the uptake of PNAs and PNA-peptide conjugates by immortal and primary human cells and compare peptide-mediated and DNA/lipid-mediated delivery strategies. We find that both peptide-mediated and lipid-mediated delivery strategies promote entry of PNA and PNA-peptide conjugates into cells. Confocal microscopy reveals a punctate distribution of PNA and PNA-peptide conjugates regardless of the delivery strategy used. Peptide D(AAKK)(4) and a peptide containing a nuclear localization sequence (NLS) promote the spontaneous delivery of antisense PNAs into cultured cells. The PNA-D(AAKK)(4) conjugate inhibits expression of human caveolin 1 (hCav-1) in both HeLa and primary endothelial cells. DNA/lipid-mediated delivery requires less PNA, while peptide-mediated delivery is simpler and is less toxic to primary cells. The ability of PNA-peptide conjugates to enter primary and immortal human cells and inhibit gene expression supports the use of PNAs as antisense agents for investigating the roles of proteins in cells. Both DNA/lipid-mediated and peptide-mediated delivery strategies are efficient, but the compartmentalized localization of PNAs suggests that improving the cellular distribution may lead to increased efficacy.

(−)-Epigallocatechin-3-O-gallate (EGCG) has useful antiviral, antimicrobial, antitoxin, and antitumor properties. Previously, Mori et al. (2008) found that addition of long acyl chains (C16–18) to EGCG enhanced its anti-influenza virus activity up to 44-fold. The chemical stability of EGCG against oxidative degradation was also enhanced by acylation. We further evaluated the in vitro activity spectrum of the EGCG derivatives against a wide range of bacteria and fungi. A series of EGCG O-acyl derivatives were synthesized by lipase-catalyzed transesterification. These derivatives exhibited several-fold higher activities than EGCG, particularly against Gram-positive organisms. Antifungal MICs of the derivatives were also two to fourfold lower than those of EGCG. The activities of the EGCG derivatives against Gram-negative bacteria were not distinguishable from those of EGCG. Among the derivatives evaluated, MICs of dioctanoate and palmitate (C16) for 17 Staphylococcus aureus strains were 4–32 μg/ml, although MIC of EGCG for these 17 strains was ≥128 μg/ml. C16 demonstrated rapid bactericidal activity against methicillin-resistant S. aureus (MRSA) ATCC43300 at ≥16 μg/ml. The enhanced activity of C16 against S. aureus was supported by its increased membrane-permeabilizing activity determined by increased SYTOX Green uptake. The EGCG derivatives were exported in Escherichia coli using the efflux pump AcrAB–TolC. The tolC deletion mutant exhibited higher sensitivity to EGCG and the derivatives than wild-type. Addition of long alkyl chains to EGCG significantly enhanced its activities against several bacteria and fungi, particularly against S. aureus including MRSA. C16 might potentially become under specified circumstances an alternative or supplement to antibiotics and disinfectants in the future.

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