Selection of RNAs that bind to duplex DNA at neutral pH

Garrett A. Soukup, Andrew D. Ellington, L. James Maher

Research output: Contribution to journalArticlepeer-review

22 Scopus citations


RNAs that are capable of binding duplex DNA in a site-specific manner have potential applications in gene therapy strategies. Such RNAs might be targeted to DNA sequences in a gene promoter and prevent initiation of transcription by occluding transcription factors and/or RNA polymerases. RNA oligonucleotides that bind homopurine/homopyrimidine DNA sequences by forming triple-helical complexes involving T·A·T and C+·G·C base-triplets can be rationally designed. However, the formation of such pyrimidine motif triple helices typically requires mildly acidic conditions. In addition, the proper oligonucleotide sequence must be optimally presented within a longer RNA transcript if it is to be synthesized in vivo. To address these issues, RNAs were selected from pools of random sequences for binding to a homopurine/homopyrimidine DNA sequence. RNAs selected for binding the duplex DNA target between pH 6.5 and pH 7.4 were characterized by sequence analysis and binding studies. All RNAs isolated by selection and amplification were found to contain a pyrimidine recognition sequence for binding the duplex DNA target via conventional triple helix formation. The selected ~85 nt RNAs have dissociation constants that approach, but do not surpass, the binding affinity of a 21 nt RNA oligonucleotide that binds the DNA target sequence by forming a canonical triple helix. The presence of a pyrimidine recognition sequence within a longer RNA transcript is not sufficient for high affinity. Experimental data and secondary structure predictions suggest that the context of the pyrimidine recognition sequence within selected RNAs is a very important determinant of DNA binding affinity. These studies provide insight into the development of RNA transcripts that may function as gene-specific repressors by forming triple helices with DNA in vivo.

Original languageEnglish (US)
Pages (from-to)216-228
Number of pages13
JournalJournal of Molecular Biology
Issue number2
StatePublished - Jun 7 1996


  • Aptamers
  • Biotin/streptavidin
  • In vitro selection
  • Pyrimidine motif
  • Triple helix formation

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Structural Biology


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