Strontium and DNA Aptamer Folding

Synonyms

d(G₂T₂G₂TGTG₂T₂G₂); G₄-DNA; G-quartet; G-tetrad; Intramolecular G-quadruplex structure; Telomere-like structure; Thrombin aptamer

Definition

In addition to classical double-stranded helical structure, some DNA sequences can form some unusual structures. Such unusual nucleic acid structures are illustrated by aptamers that consist of short synthetic DNA or RNA sequences that adapt well-defined three-dimensional structures and bind to specific biomolecules. For example, the 15-mer oligonucleotide with a d(G₂T₂G₂TGTG₂T₂G₂) sequence is one of the thrombin-binding aptamers (TBA) that binds to the serine protease thrombin with high affinity and inhibits the thrombin-catalyzed fibrin-clot formation. The unique spatial structure of TBA, the intramolecular G-quadruplex, is stabilized by the specific coordination of metal ions. Although various cations can stabilize aptamers, the highest stability was ascribed to Sr²⁺-aptamers.

Unusual DNA Structures: Telomeres, G-Quadruplexes, and Aptamers

Among various unusual DNA structures are telomeres, which are repetitive nucleotide sequences located at the termini of linear chromosome. Telomeres are known to stabilize the ends of chromosomes (Blackburn 1991a, b), protecting them from deterioration or from fusion with neighboring chromosomes, and control the proper replication and segregation of eukaryotic chromosomes (DePamphilis 1993). In fact, since the eukaryotic DNA replication enzymes cannot replicate the DNA sequences present at the ends of the chromosomes, these sequences and the information they carry may get lost. The telomeres, that “cap” the end-sequences, serve as disposable buffers at the ends of the chromosomes, get lost in the process of DNA replication, being consumed during cell division, and are replenished by an enzyme, telomerase reverse transcriptase.

Telomere length varies greatly between species, ranging from ∼300 base pairs in yeast to many kilobases in humans and is typically composed of arrays of guanine-rich, six- to eight-base-pair-long repeats. The telomere ends of 12–16 bases usually have G and T repeats in one strand that overhangs at the 3′ end, which is essential for telomere maintenance and capping (Henderson et al. 1987; Henderson and Blackburn 1989). Telomeres are known to form large loop structures, telomere loops (T-loops), where the single-stranded DNA curls around in a long circle stabilized by telomere-binding proteins (Griffith et al. 1999). At the very end of the T-loop, the single-stranded telomere DNA is held onto a region of double-stranded DNA by the telomere strand disrupting the double-helical DNA, base pairing to one of the two strands, thus forming a specific triple-stranded structure, displacement loop or d-loop (Burge et al. 2006).

Nucleic acid sequences enriched in guanine are capable of forming a four-stranded structure, G-quadruplexes, also known as G-quartets, G-tetrads, or G₄-DNA, which can be formed by DNA and RNA, be parallel or antiparallel (depending on the direction of the strands or parts of a strand that form the tetrads), and can represent intramolecular, bimolecular, or tetramolecular structures. Since telomeres in all vertebrates consist of many repeats of the d(GGTTAG) sequence, they can form G-quadruplexes. In the G-tetrad structure, four guanine bases associate through Hoogsteen hydrogen bonding to form a square planar structure called a guanine tetrad, and two or more guanine tetrads can stack on top of each other to form a G-quadruplex. The quadruplex structure is further stabilized by the presence of a cation, such as Na⁺ or K⁺, which sits in a central channel between each pair of tetrads (see Fig. 1) (Williamson et al. 1989; Miura et al. 1995).

Strontium and DNA Aptamer Folding, Fig. 1

Structure of a G-quadruplex. (a) A G-tetrad. (b) An intramolecular G-quadruplex. (c) Structure of parallel quadruplexes that can be formed by human telomeric DNA (PDB ID: 1KF1)

Besides being found in telomeres, G-quadruplexes can be found in promoter regions of some genes (Simonsson et al. 1998; Siddiqui-Jain et al. 2002). Quadruplex folding rules have been elaborated and used for the genome-wide analysis of the abundance of quadruplexes. These analyses revealed that human genome contains 376,000 Putative Quadruplex Sequences (Huppert and Balasubramanian 2005). Furthermore, quadruplexes are also common in the bacterial genomes, where they are predominantly located within promoters of genes pertaining to transcription, secondary metabolite biosynthesis, and signal transduction (Rawal et al. 2006).

G- quadruplexes are also commonly found in DNA aptamers, which have been identified by some selection process to bind specific targets (Ellington and Szostak 1990). The remarkable kinetic and thermodynamic stability of G-quadruplexes is determined by the tight association of cations with guanine residues (Kang et al. 1992).

Strontium and Thrombin Aptamers

The ability of nucleic acids to fold into a wide array of different structures was utilized in the development of techniques for the generation of aptamers (Ellington and Szostak 1990). Aptamers are DNA or RNA oligonucleotides that have been screened from a randomly generated population of sequences for their ability to bind a desired molecular target (Ellington and Szostak 1990).

The aptamer isolation process consists of repeated cycles of selection for, and enrichment of, oligonucleotides with an affinity to a specific target (e.g., with the ability to inhibit the activity of a target protein), followed by the PCR-based amplification of these sequences. One of the most studies aptamers is the thrombin-binding aptamer (TBA) selected for its ability to bind to thrombin and inhibit thrombin-catalyzed fibrin-clot formation in vitro (Bock et al. 1992). TBA is the d(G₂T₂G₂TGTG₂T₂G₂) oligonucleotide, which is able to form a complex structure that is comprised of two G-quartets connected by a TGT loop at the center and two T₂ loops (see Fig. 2), and stabilized by the formation of an intramolecular complex with K⁺ (Macaya et al. 1993).

Strontium and DNA Aptamer Folding, Fig. 2

Structure of the thrombin aptamer. (a) Sequence of the thrombin aptamer. (b) Schematic structure of TBA. (c) TBA structure stabilized by interaction with Sr²⁺ (PDBID: 1RDE)

Detailed structural characterization revealed that K⁺, Rb⁺, NH ⁺₄ , Sr²⁺, and Ba²⁺ are able to form stable intramolecular cation − aptamer complexes at temperatures above 25°C, whereas the cations Li⁺, Na⁺, Cs⁺, Mg²⁺, and Ca²⁺ were shown to form weaker complexes at very low temperatures (Kankia and Marky 2001). Rationalization for these observations is based on the consideration of ionic radii. Here, metal cations with an ionic radius in the range 1.3 − 1.5 Å fit well within the two G-quartets of the complex, whereas the other cations cannot (Kankia and Marky 2001). Comparison of the G-quadruplexes with K⁺ and Sr²⁺ revealed that the Sr²⁺ aptamer complex unfolds with a higher transition temperature and lower endothermic heat, but its favorable formation (in terms of ΔG°) is comparable to that of the K⁺ aptamer complex. Furthermore, hydration/dehydration plays a similar role in the formation of both K⁺ and Sr²⁺ aptamers, where there are two major hydration-related contributions, dehydration of both cations and guanine O6 atomic groups and the water uptake upon folding of a single strand into a G-quadruplex structure (Kankia and Marky 2001).

Cross-References

Calcium, Physical and Chemical Properties

Strontium, Physical and Chemical Properties