Four base recognition by triplex-forming oligonucleotides at physiological pH

We have achieved recognition of all 4 bp by triple helix formation at physiological pH, using triplex-forming oligonucleotides that contain four different synthetic nucleotides. BAU [2′-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine] recognizes AT base pairs with high affinity, MeP (3-methyl-2 aminopyridine) binds to GC at higher pHs than cytosine, while APP (6-(3-aminopropyl)-7-methyl-3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one) and S [N-(4-(3-acetamidophenyl)thiazol-2-yl-acetamide)] bind to CG and TA base pairs, respectively. Fluorescence melting and DNase I footprinting demonstrate successful triplex formation at a 19mer oligopurine sequence that contains two CG and two TA interruptions. The complexes are pH dependent, but are still stable at pH 7.0. BAU, MeP and APP retain considerable selectivity, and single base pair changes opposite these residues cause a large reduction in affinity. In contrast, S is less selective and tolerates CG pairs as well as TA.

[1]  M. Rots,et al.  Therapeutic modulation of endogenous gene function by agents with designed DNA-sequence specificities. , 2003, Nucleic acids research.

[2]  F. J. Luque,et al.  Nucleic Acid Triple Helices: Stability Effects of Nucleobase Modifications , 2002 .

[3]  D. Loakes,et al.  Survey and summary: The applications of universal DNA base analogues. , 2001, Nucleic acids research.

[4]  A. Khorlin,et al.  Correction of chromosomal point mutations in human cells with bifunctional oligonucleotides , 1999, Nature Biotechnology.

[5]  C. Leumann,et al.  5-Substituted 2-Aminopyridine C-Nucleosides as Protonated Cytidine Equivalents: Increasing Efficiency and Selectivity in DNA Triple-Helix Formation , 1997 .

[6]  S. Neidle,et al.  Efficient triple helix formation by oligodeoxyribonucleotides containing α-or β2-amino-5-( 2-deoxy-D-ribofuranosyl ) pyridine residues , 1996 .

[7]  A. Weis,et al.  Elucidation of the sequence-specific third-strand recognition of four Watson-Crick base pairs in a pyrimidine triple-helix motif: T.AT, C.GC, T.CG, and G.TA. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Sabrina Buchini Dipl.-Chem. and,et al.  Stable and Selective Recognition of Three Base Pairs in the Parallel Triple-Helical DNA Binding Motif† , 2004 .

[9]  S. Benner,et al.  pH-Independent Triple Helix Formation by an Oligonucleotide Containing a Pyrazine Donor-Donor-Acceptor Base , 1995 .

[10]  David A Rusling,et al.  Stable recognition of TA interruptions by triplex forming oligonucleotides containing a novel nucleoside. , 2005, Biochemistry.

[11]  D. Gowers,et al.  Towards mixed sequence recognition by triple helix formation. , 1999, Nucleic acids research.

[12]  F. Natt,et al.  Dual Recognition of Double-Stranded DNA by 2'-Aminoethoxy-Modified Oligonucleotides. , 1998, Angewandte Chemie.

[13]  J. S. Lee,et al.  Poly(pyrimidine) . poly(purine) synthetic DNAs containing 5-methylcytosine form stable triplexes at neutral pH. , 1984, Nucleic acids research.

[14]  V. Potaman.  Applications of triple-stranded nucleic acid structures to DNA purification, detection and analysis , 2003, Expert review of molecular diagnostics.

[15]  P. Nielsen,et al.  A NOVEL PEPTIDE NUCLEIC ACID MONOMER FOR RECOGNITION OF THYMINE IN TRIPLE-HELIX STRUCTURES , 1997 .

[16]  R. Benhida,et al.  Incorporation of a novel nucleobase allows stable oligonucleotide-directed triple helix formation at the target sequence containing a purine.pyrimidine interruption. , 2001, Chemical communications.

[17]  Andrea B. Staubli,et al.  Sequence specificity of the non-natural pyrido[2, 3-d]pyrimidine nucleoside in triple helix formation , 1994, Nucleic Acids Res..

[18]  Y. Hari,et al.  Triplex formation involving 2',4'-BNA with 2-pyridone base analogue: efficient and selective recognition of C:G interruption. , 2001, Nucleic acids research. Supplement.

[19]  C. Leumann,et al.  Stable and selective recognition of three base pairs in the parallel triple-helical DNA binding motif. , 2004, Angewandte Chemie.

[20]  Quantitative footprinting analysis of drug‐DNA interactions: Fe (III) methidium‐propyl‐EDTA as a probe , 1989, Electrophoresis.

[21]  J. Toulmé,et al.  Specific regulation of gene expression by antisense, sense and antigene nucleic acids. , 1990, Biochimica et biophysica acta.

[22]  F. Nagatsugi,et al.  Selective formation of stable triplexes including a TA or a CG interrupting site with new bicyclic nucleoside analogues (WNA). , 2004, Journal of the American Chemical Society.

[23]  D. Gowers,et al.  DNA triple helix formation at oligopurine sites containing multiple contiguous pyrimidines. , 1997, Nucleic acids research.

[24]  J. Toulmé,et al.  Quinazoline-2,4(1H,3H)-dione as a substitute for thymine in triple-helix forming oligonucleotides: a reassessment. , 1996, Nucleic acids research.

[25]  P. Dervan,et al.  Recognition of thymine adenine.base pairs by guanine in a pyrimidine triple helix motif. , 1989, Science.

[26]  P. Dervan,et al.  Design of an N7-Glycosylated Purine Nucleoside for Recognition of GC Base Pairs by Triple Helix Formation , 1995 .

[27]  P. Miller,et al.  Recognition of a guanine-cytosine base pair by 8-oxoadenine. , 1992, Biochemistry.

[28]  David A Rusling,et al.  Selectivity and affinity of triplex-forming oligonucleotides containing 2'-aminoethoxy-5-(3-aminoprop-1-ynyl)uridine for recognizing AT base pairs in duplex DNA. , 2004, Nucleic acids research.

[29]  P. Dervan,et al.  Design of a Nonnatural Deoxyribonucleoside for Recognition of GC Base Pairs by Oligonucleotide‐Directed Triple Helix Formation. , 1992 .

[30]  J. Feigon,et al.  The Nonnatural Deoxyribonucleoside D3 Incorporated in an Intramolecular DNA Triplex Binds Sequence Specifically by Intercalation , 1993 .

[31]  Y. Hari,et al.  Triplex formation involving 2',4'-BNA with isoquinolone base analogue: efficient and selective recognition of C:G interruption. , 2002, Nucleic acids research. Supplement.

[32]  Tom Brown,et al.  High throughput measurement of duplex, triplex and quadruplex melting curves using molecular beacons and a LightCycler. , 2002, Nucleic acids research.

[33]  T. Brown,et al.  5-(1-propargylamino)-2'-deoxyuridine (UP): a novel thymidine analogue for generating DNA triplexes with increased stability. , 1999, Nucleic acids research.

[34]  S. Neidle,et al.  Recognition of GC base pairs by triplex forming oligonucleotides containing nucleosides derived from 2-aminopyridine. , 1997, Nucleic acids research.

[35]  B. Cuenoud,et al.  Importance of clustered 2'-O-(2-aminoethyl) residues for the gene targeting activity of triple helix-forming oligonucleotides. , 2004, Biochemistry.

[36]  K. G. Rajeev,et al.  Triplex formation at physiological pH: comparative studies on DNA triplexes containing 5-Me-dC tethered at N4 with spermine and tetraethyleneoxyamine. , 1997, Nucleic acids research.

[37]  Chin-Yi Huang,et al.  Triplex formation by oligonucleotides containing novel deoxycytidine derivatives [published erratum appears in Nucleic Acids Res 1997 Sep 15;25(18): following 3750] , 1996, Nucleic Acids Res..

[38]  P. Dervan,et al.  Effects of an abasic site on triple helix formation characterized by affinity cleaving. , 1991, Nucleic Acids Research.

[39]  W. Soussou,et al.  A NEW PYRIMIDINE NUCLEOSIDE (M5OXC) FOR THE PH-INDEPENDANT RECOGNITION OF G-C BASE PAIRS BY OLIGONUCLEOTIDE-DIRECTED TRIPLEX FORMATION , 1994 .

[40]  C. Leumann,et al.  Recognition of a G-C base pair by α-N7-deoxyinosine within the pyrimidine-purine-pyrimidine DNA triple helical motif , 1996 .

[41]  K R Fox,et al.  Targeting DNA with triplexes. , 2000, Current medicinal chemistry.

[42]  C. Leumann,et al.  Recent improvements in antigene technology. , 2003, Current opinion in chemical biology.

[43]  C. Leumann,et al.  Selective recognition of a C-G base-pair in the parallel DNA triple-helical binding motif. , 1999, Bioorganic & medicinal chemistry letters.

[44]  C. Leumann,et al.  Dual recognition of a C-G pyrimidine-purine inversion site: synthesis and binding properties of triplex forming oligonucleotides containing 2'-aminoethoxy-5-methyl-1H-pyrimidin-2-one ribonucleosides , 2003 .

[45]  L. Kan,et al.  Triplex formation of oligonucleotides containing 2'-O-methylpseudoisocytidine in substitution for 2'-deoxycytidine , 1991 .

[46]  S. Zimmerman,et al.  Synthesis and duplex DNA recognition studies of oligonucleotides containing a ureido isoindolin-1-one homo-N-nucleoside. A comparison of host-guest and DNA recognition studies. , 2004, Bioorganic & medicinal chemistry.

[47]  P. Glazer,et al.  Specific mutations induced by triplex-forming oligonucleotides in mice. , 2000, Science.

[48]  S. Neidle,et al.  Efficient triple helix formation by oligodeoxyribonucleotides containing alpha- or beta-2-amino-5-(2-deoxy-D-ribofuranosyl) pyridine residues. , 1996, Nucleic acids research.

[49]  L. Marky,et al.  Triple helix forming TRIPside molecules that target mixed purine/pyrimidine DNA sequences. , 2004, Biochemistry.

[50]  David A Rusling,et al.  Recognition of CG inversions in DNA triple helices by methylated 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleoside analogues. , 2005, Chemical communications.

[51]  S. Napoli,et al.  DNA binding and antigene activity of a daunomycin-conjugated triplex-forming oligonucleotide targeting the P2 promoter of the human c-myc gene. , 2004, Nucleic acids research.

[52]  B. Cuenoud,et al.  Stable DNA triple helix formation using oligonucleotides containing 2'-aminoethoxy,5-propargylamino-U. , 2002, Biochemistry.

[53]  D. Barawkar,et al.  Triplex formation at physiological pH by oligonucleotides incorporating 5-Me-dC-(N4-spermine). , 1994, Biochemical and biophysical research communications.