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.