Twisted intercalating nucleic acid (TINA) is a

nucleic acid Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...

molecule that, when added to triplex-forming

oligonucleotide Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids c ...

s (TFOs), stabilizes Hoogsteen triplex DNA formation from double-stranded DNA (dsDNA) and TFOs. Its ability to twist around a triple bond increases ease of intercalation within double stranded DNA in order to form triplex DNA. Certain configurations have been shown to stabilize Watson-Crick antiparallel duplex DNA. TINA-DNA primers have been shown to increase the specificity of binding in PCR. The use of TINA insertions in G-quadruplexes has also been shown to enhance anti-HIV-1 activity. TINA stabilized PT demonstrates improved sensitivity and specificity of DNA based clinical diagnostic

assays An assay is an investigative (analytic) procedure in laboratory medicine, mining, pharmacology, environmental biology and molecular biology for qualitatively assessing or quantitatively measuring the presence, amount, or functional activity of a ...

Triplex DNA

Triple helixes are formed when a single-stranded triplex-forming

(TFO) binds to a

purine Purine is a heterocyclic compound, heterocyclic aromatic organic compound that consists of two rings (pyrimidine and imidazole) fused together. It is water-soluble. Purine also gives its name to the wider class of molecules, purines, which includ ...

-containing strand of dsDNA through specific major groove interactions. Generally, the third-strand affinity of a TFO is low, due to the requirement for the formation of pH-sensitive C+–G–C Hoogsteen base triplexes under physiological conditions in the parallel (

pyrimidine Pyrimidine (; ) is an aromatic, heterocyclic, organic compound similar to pyridine (). One of the three diazines (six-membered heterocyclics with two nitrogen atoms in the ring), it has nitrogen atoms at positions 1 and 3 in the ring. The other ...

) binding motif. Modification of TFOs has been attempted in order to improve their binding affinities to their targets and to lessen restrictions in the dsDNA sequence with the design of new triplex

nucleobases Nucleobases, also known as ''nitrogenous bases'' or often simply ''bases'', are nitrogen-containing biological compounds that form nucleosides, which, in turn, are components of nucleotides, with all of these monomers constituting the basic b ...

. Recently, it has been found that bulge insertions of (R)-1-O- -(1-pyrenylethynyl)phenylmethyllycerol (TINA) into the middle of homopyrimidine oligodeoxynucleotides can give rise to thermal stability in Hoogsteen-type triplexes and duplexes, whereas Watson–Crick-type duplexes of the same nucleotide content were destabilized. To increase ∆Tm, base mismatches should be placed in the center of the TFO and when feasible, A, C or T to G base mismatches should be avoided. Base mismatches can be neutralized by intercalation of a TINA on each side of the base mismatch and masked by a TINA intercalating direct 3'or 5' of it.

Applications

Assay specificity

Diagnostic assays using DNA hybridization are limited by the dissociation of antiparallell duplex helices. This can be improved by using DNA stabilizing molecules such as intercalators like ortho-TINA, which will stabilize the duplex formation. Studies show that the greatest increase in stability occurred when intercalating primers were used at the 3’ and 5’ ends. Placement of a TINA molecule in the oligonucleotide is capable of improving the analytical sensitivity of the probe hybridization. Para-TINA molecules decreases Tm in all positions especially when at the center of the oligonucleotide, while in the ortho-TINA molecules, the improvement was seen anywhere with neutralization at the center. Combination of terminal para- or ortho- molecule with an internal TINA molecule showed the highest increase of Tm. TINA molecules should be placed terminally for maximum increase in Tm. An increase in Tm increases the specificity of assays, like PCR.

Anti-HIV-1 activity

Recent studies show that the use of TINA insertions in G-quadruplexes has also been shown to enhance anti-HIV-1 activity. In such studies, two G quadruplexes forming sequences which exhibit anti-HIV-1 activity on cell lines were modified using

locked nucleic acid A locked nucleic acid (LNA), also known as bridged nucleic acid (BNA), and often referred to as inaccessible RNA, is a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon. Th ...

(LNA) or insertions of TINA. Incorporation of this provides as much as 8-fold improvement of anti-HIV-1 activity and the introduction of 5’ phosphate was shown to inhibit the dimerization of G-quadruplex. Many

antiviral Antiviral drugs are a class of medication used for treating viral infections. Most antivirals target specific viruses, while a broad-spectrum antiviral is effective against a wide range of viruses. Unlike most antibiotics, antiviral drugs do no ...

quadruplexes forming oligonucleotides formed more thermally stable G-quadruplexes and also high-order G quadruplex structures, which may be responsible for antiviral activity observed.

Therapeutic applications

TFOs hold promise in anti-gene therapy, due to their high sequence specificity. However, the

potassium Potassium is the chemical element with the symbol K (from Neo-Latin ''kalium'') and atomic number19. Potassium is a silvery-white metal that is soft enough to be cut with a knife with little force. Potassium metal reacts rapidly with atmosphe ...

levels

in vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...

promote TFOs to form G-quartet structures singularly, preventing the TFOS from interacting in a triplex formation and decreasing the effectiveness TFO cell therapies. However, as shown by Paramasivam et al., bulge insertions of (R)-1-O- -(1-pyrenylethynyl)phenylmethyllycerol (TINA) into TFOs with high

guanine Guanine () ( symbol G or Gua) is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine (uracil in RNA). In DNA, guanine is paired with cytosine. The guanine nucleoside is called ...

concentrations greatly decreases the presence of self-association via potassium. TINA-TFOs, then, may be used in the future to target the

genome In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...

in vivo and perform genome manipulation toward a therapeutic end. The use of purine TINA-TFOs holds specific promise as antigene molecules toward the KRAS proto-

oncogene An oncogene is a gene that has the potential to cause cancer. In tumor cells, these genes are often mutated, or expressed at high levels.

References

{{reflist Nucleic acids