The detrimental effect of orotic acid substitution in the peptide nucleic acid strand on the stability of PNA2:NA triple helices

We have investigated the incorporation of C6 derivatives of uracil into polypyrimidine peptide nucleic acid oligomers. Starting with uracil-6-carboxylic acid (orotic acid), a peptide nucleic acid monomer compatible with Fmoc-based synthesis was prepared. This monomer then served as a convertible nucleobase whereupon treatment of the resin-bound methyl orotate containing hexamers with hydroxide or amines cleanly converted the ester to an orotic acid or orotamide-containing peptide nucleic acid. Peptide nucleic acid hexamers containing the C6-modified nucleobase hybridized to both poly(riboadenylic acid) and poly(deoxyriboadenylic acid) via triplex formation. Complexes formed with poly(riboadenylic acid) were more stable than those formed with poly(dexoyriboadenylic acid), as measured by temperature-dependent UV spectroscopy. However, both of these complexes were destabilized relative to the complexes formed by an unmodified peptide nucleic acid oligomers. Internal or doubly substituted hexamers are destabilized more strongly than a terminally substituted one, and the type of substitution (carboxamide, ester, carboxylic acid) affects the overall triplex stability. These results clearly show that incorporation of a C6-substituted uracil into polypyrimidine PNA is detrimental to triplex formation. We have also extended this chemistry to incorporate uracil-5-methylcarboxylate into a peptide nucleic acid hexamer. After on-resin conversion of the C5 ester to the 3-(N,N-dimethylamino)propylamide, significant stabilization of the triplex formed with poly(riboadenylic acid) was observed, which illustrates the compatibility of C5 substitution with peptide nucleic acid directed triple helix formation. Key words: peptide nucleic acid, triple helix, orotic acid, orotamide, PNA.

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Stability of an RNA•DNA–DNA triple helix depends on base triplet composition and length of the RNA third strand

Nucleic Acids Research ◽

10.1093/nar/gkz573 ◽

2019 ◽

Vol 47 (14) ◽

pp. 7213-7222 ◽

Cited By ~ 4

Author(s):

Charlotte N Kunkler ◽

Jacob P Hulewicz ◽

Sarah C Hickman ◽

Matthew C Wang ◽

Phillip J McCown ◽

...

Keyword(s):

Relative Stability ◽

Triple Helix ◽

Multiple Factors ◽

Dna And Rna ◽

Helix Formation ◽

Canonical Base ◽

Triple Helix Formation ◽

Triple Helices ◽

Dna Triple Helix ◽

The Stability

AbstractRecent studies suggest noncoding RNAs interact with genomic DNA, forming an RNA•DNA–DNA triple helix that regulates gene expression. However, base triplet composition of pyrimidine motif RNA•DNA–DNA triple helices is not well understood beyond the canonical U•A–T and C•G–C base triplets. Using native gel-shift assays, the relative stability of 16 different base triplets at a single position, Z•X–Y (where Z = C, U, A, G and X–Y = A–T, G–C, T–A, C–G), in an RNA•DNA–DNA triple helix was determined. The canonical U•A–T and C•G–C base triplets were the most stable, while three non-canonical base triplets completely disrupted triple-helix formation. We further show that our RNA•DNA–DNA triple helix can tolerate up to two consecutive non-canonical A•G–C base triplets. Additionally, the RNA third strand must be at least 19 nucleotides to form an RNA•DNA–DNA triple helix but increasing the length to 27 nucleotides does not increase stability. The relative stability of 16 different base triplets in DNA•DNA–DNA and RNA•RNA–RNA triple helices was distinctly different from those in RNA•DNA–DNA triple helices, showing that base triplet stability depends on strand composition being DNA and/or RNA. Multiple factors influence the stability of triple helices, emphasizing the importance of experimentally validating formation of computationally predicted triple helices.

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Highly stable triple helix formation by homopyrimidine (l)-acyclic threoninol nucleic acids with single stranded DNA and RNA

Organic & Biomolecular Chemistry ◽

10.1039/c4ob02328e ◽

2015 ◽

Vol 13 (8) ◽

pp. 2366-2374 ◽

Cited By ~ 4

Author(s):

Vipin Kumar ◽

Venkitasamy Kesavan ◽

Kurt V. Gothelf

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Triple Helix ◽

Helical Structures ◽

Single Stranded Dna ◽

Dna And Rna ◽

Helix Formation ◽

Triple Helix Formation

Homopyrimidine acyclic (l)-threoninol nucleic acid (aTNA) was synthesized and found to form highly stable (l)-aTNA–DNA–(l)-aTNA and (l)-aTNA–RNA–(l)-aTNA triple helical structures.

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Targeted mutagenesis in mammalian cells mediated by intracellular triple helix formation.

Molecular and Cellular Biology ◽

10.1128/mcb.15.3.1759 ◽

1995 ◽

Vol 15 (3) ◽

pp. 1759-1768 ◽

Cited By ~ 118

Author(s):

G Wang ◽

D D Levy ◽

M M Seidman ◽

P M Glazer

Keyword(s):

Mammalian Cells ◽

Triple Helix ◽

Uv Light ◽

Simian Virus 40 ◽

Target Site ◽

Targeted Mutagenesis ◽

Cos Cells ◽

Triplex Formation ◽

Helix Formation ◽

Triple Helix Formation

As an alternative to standard gene transfer techniques for genetic manipulation, we have investigated the use of triple helix-forming oligonucleotides to target mutations to selected genes within mammalian cells. By treating monkey COS cells with oligonucleotides linked to psoralen, we have generated targeted mutations in a simian virus 40 (SV40) vector contained within the cells via intracellular triple helix formation. Oligonucleotide entry into the cells and sequence-specific triplex formation within the SV40 DNA deliver the psoralen to the targeted site. Photoactivation of the psoralen by long-wavelength UV light yields adducts and thereby mutations at that site. We engineered into the SV40 vector novel supF mutation reporter genes containing modified polypurine sites amenable to triplex formation. By comparing the abilities of a series of oligonucleotides to target these new sites, we show that targeted mutagenesis in vivo depends on the strength and specificity of the third-strand binding. Oligonucleotides with weak target site binding affinity or with only partial target site homology were ineffective at inducing mutations in the SV40 vectors within the COS cells. We also show that the targeted mutagenesis is dependent on the oligonucleotide concentration and is influenced by the timing of the oligonucleotide treatment and of the UV irradiation of the cells. Frequencies of intracellular targeted mutagenesis in the range of 1 to 2% were observed, depending upon the conditions of the experiment. DNA sequence analysis revealed that most of the mutations were T.A-to-A.T transversions precisely at the targeted psoralen intercalation site. Several deletions encompassing that site were also seen. The ability to target mutations to selected sites within mammalian cells by using modified triplex-forming oligonucleotides may provide a new research tool and may eventually lead to therapeutic applications.

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Effect of prevention of procollagen triple-helix formation on proline 3-hydroxylation in freshly isolated chick-embryo tendon cells

Biochemical Journal ◽

10.1042/bj1960203 ◽

1981 ◽

Vol 196 (1) ◽

pp. 203-206 ◽

Cited By ~ 10

Author(s):

K Majamaa

Keyword(s):

Carboxylic Acid ◽

Chick Embryo ◽

Triple Helix ◽

Hydroxylase Activity ◽

Intact Cells ◽

Helix Formation ◽

Tendon Cells ◽

Triple Helix Formation

Inhibition of procollagen triple-helix formation by the addition of cis-hydroxyproline or azetidine-2-carboxylic acid increased the synthesis of 3-hydroxy[14C]proline 1.7-1.8-fold in pulse-chase experiments with freshly isolated chick-embryo tendon cells. The amount of 3-hydroxy[14C]proline, expressed as a percentage of the total 14C radioactivity in hydroxyproline, reached 8.4%. Control experiments indicated that the two analogues had no effect on the prolyl 3-hydroxylase activity of these cells. The data suggest that the time available before triple-helix formation in part regulates the extent of the 3-hydroxylation of proline in the biosynthesis of collagen in intact cells.

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