The present invention describes a polymer composition effective to bind, in a sequence-specific manner to a target sequence of a duplex polynucleotide, at least two different-oriented Watson/Crick base-pairs at selected positions in the target sequence. The composition includes an uncharged backbone with 5- or 6-membered cyclic backbone structures and selected bases attached to the backbone structures effective to hydrogen bond specifically with different oriented base-pairs in the target sequence. Also disclosed are subunits useful for the construction of the polymer composition. The present invention further includes methods for (i) coupling a first free or polymer-terminal subunit, and (ii) isolating, from a liquid sample, a target duplex nucleic acid fragment having a selected sequence of base-pairs.

Sequence-specific binding polymers for duplex nucleic acids

The binding of a variety of deoxyribo and ribo homo- and copolynucleotide complementary duplexes to agarose-linked homopolynucleotide affinity columns has been studied. The results provide information concerning the specificity of recognition of complementary base pairs of nucleic acids through a mechanism that involves triple-helix formation under physiological conditions of ionic strength, pH, and temperature. The method employed made it possible, for the first time, to survey the full range of base triplets conceivable from the canonical nucleic acid bases and, in addition, hypoxanthine and thereby to differentiate between those triplets which can and cannot form. Certain previously observed features of the stereochemistry of double-helical targets for third-strand binding are confirmed, and some unrecognized features are elaborated. These include a general requirement for clusters of purine residues in one strand, protonation of third-strand C residues, the ability of natural third-strand residues to distinguish between A.T/U and G.C base pairs, and a capacity of third-strand (unnatural) I residues to recognize all base pairs within such clusters. Thus, the basis for a third-strand binding code is demonstrated.

Specificity in formation of triple-stranded nucleic acid helical complexes: studies with agarose-linked polyribonucleotide affinity columns.

It has been nearly 63 years since the first characterization of an RNA triple helix in vitro by Gary Felsenfeld, David Davies, and Alexander Rich. An RNA triple helix consists of three strands: A Watson-Crick RNA double helix whose major-groove establishes hydrogen bonds with the so-called "third strand". In the past 15 years, it has been recognized that these major-groove RNA triple helices, like single-stranded and double-stranded RNA, also mediate prominent biological roles inside cells. Thus far, these triple helices are known to mediate catalysis during telomere synthesis and RNA splicing, bind to ligands and ions so that metabolite-sensing riboswitches can regulate gene expression, and provide a clever strategy to protect the 3' end of RNA from degradation. Because RNA triple helices play important roles in biology, there is a renewed interest in better understanding the fundamental properties of RNA triple helices and developing methods for their high-throughput discovery. This review provides an overview of the fundamental biochemical and structural properties of major-groove RNA triple helices, summarizes the structure and function of naturally occurring RNA triple helices, and describes prospective strategies to isolate RNA triple helices as a means to establish the "triplexome". This article is categorized under: RNA Structure and Dynamics > RNA Structure and Dynamics RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.

Unraveling the structure and biological functions of RNA triple helices.

The nucleotide sequence of the second part of the simian virus 40 DNA HindII + III restriction fragment A is presented. The sequence extends from map position 0.533 to 0.424 and together with the first part of Hind-A [Volckaert et al. Proc. Natl Acad. Sci. U.S.A. 75, 2160–2164 (1978)] completes the total Hind-A sequence, comprising 1169 base pairs. The second half of Hind-A includes the region corresponding to the second splicing boundary common to small tumor antigen (small-t) and large tumor antigen (large-T) mRNA and it contains coding information for an internal portion of large-T antigen. Two similar secondary structures of reasonable thermodynamic stability can be proposed for the nucleotide sequence of the pre-mRNA corresponding to the region reported here. Their possible relevance to the splicing of the SV40 early mRNAs is discussed.



The deduced amino acid sequence is 188 residues long and contains a Lys-Lys-Lys-Arg-Lys stretch which may be involved in the DNA binding capacity of large-T. A presumptive phosphorylation site is also present.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1980.tb06008.x

Nucleotide Sequence of the Simian Virus 40 HindII + III Restriction Fragment A (Second Part of the T Antigen Gene)

Duplex DNA containing oligo(dG.dC)-rich clusters can be isolated by specific binding to poly(rC)-Sephadex. This binding, probably mediated by the formation of an oligo(dG.dC)rC+ triple helix, is optimal at pH 5 in 50% formamide, 2 M LiCl; the bound DNA is recovered by elution at pH 7.5. Using this method we find that the viral DNAs PM2, lambda and SV40 contain at least 1, 1 and 2 sites for binding to poly(rC)-Sephadex, respectively. These binding sites have been mapped in the case of SV40; the binding sites can in turn be used for physical mapping studies of DNAs containing (dG.dC) clusters. Inspection of the sequence of the bound fragments of SV40 DNA shows that a (dG.dC)6-7 tract is required for the binding of duplex DNA to poly(rC)-Sephadex. Although about 60% of rabbit DNA cleaved with restriction endonuclease KpnI binds to poly(rC)-Sephadex, no binding is observed for the 5.1 kb DNA fragment generated by KpnI digestion, which contains the rabbit beta-globin gene. This indicates that oligo(dG.dC) clusters are not found close to the rabbit beta-globin gene.

F.M. van den Berg

Papers

The isolation of duplex DNA fragments containing (dG.dC) clusters by chromatography on poly(rC)-Sephadex