Recent developments show encouraging results for the use of DNA as a construction material for nanometer-sized objects. Today, however, DNA-based molecular nanoarchitectures are constructed with mainly unmodified or at best end-modified oligonucleotides, thus shifting the development of functionalized DNA structures into the limelight. One of most recent developments in this direction is the substitution of the canonical Watson-Crick base pairs by metal complexes. In this way "metal-base pairs" are created, which could potentially impart magnetic or conductive properties to DNA-based nanostructures. This review summarizes research which started almost 45 years ago with the investigation of how metal ions interact with unmodified DNA and which recently culminated in the development of artificial ligand-like nucleobases so far able to coordinate up to ten metal ions inside a single DNA duplex in a programmable fashion.

DNA--metal base pairs.

Quantification of the sixth DNA base hydroxymethylcytosine in the brain.

Aptamer therapeutics advance.

Aptamers are short DNA/RNA oligonucleotides capable of binding to target molecules with high affinity and specificity. The process of selecting an aptamer is called Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Thanks to the inherit merits, aptamers have been used in a wide range of applications, including disease diagnosis, targeted delivery agents and therapeutic uses. To date, great achievements regarding the selection, modifications and application of aptamers have been made. However, few aptamer-based products have already successfully entered into clinical and industrial use. Besides, it is still a challenge to obtain aptamers with high affinity in a more efficient way. Thus, it is important to comprehensively review the current shortage and achievement of aptamer-related technology. In this review, we first present the limitations and notable advances of aptamer selection. Then, we compare the different methods used in the kinetic characterization of aptamers. We also discuss the impetus and developments of the clinical application of aptamers.

Recent Advances in SELEX Technology and Aptamer Applications in Biomedicine

Over the last decade, many catalytically active DNA molecules (deoxyribozymes; DNA enzymes) have been identified by in vitro selection from randomsequence DNA pools. This article focuses on deoxyribozymes that cleave RNA substrates. The first DNA enzyme was reported in 1994 and cleaves an RNA linkage. Since that time, many other RNA-cleaving deoxyribozymes have been identified. Most but not all of these deoxyribozymes require a divalent metal ion cofactor such as Mg 21 to catalyze attack by a specific RNA 2 0 -hydroxyl group on the adjacent phosphodiester linkage, forming a 2 0 ,3 0 -cyclic phosphate and a 5 0 -hydroxyl group. Several deoxyribozymes that cleave RNA have utility for in vitro RNA biochemistry. Some DNA enzymes have been applied in vivo to degrade mRNAs, and others have been engineered into sensors. The practical impact of RNA-cleaving deoxyribozymes should continue to increase as additional applications are developed.

SURVEY AND SUMMARY In vitro selection, characterization, and application of deoxyribozymes that cleave RNA

We synthesized C5-modified analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate and investigated them as substrates for PCRs using Taq, Tth, Vent(exo-), KOD Dash and KOD(exo-) polymerases and pUC 18 plasmid DNA as a template. These assays were performed on two different amplifying regions of pUC18 with different T/C contents that are expected to have relatively high barriers for incorporation of either modified dU or dC. On the basis of 260 different assays (26 modified triphosphates x 5 DNA polymerases x 2 amplifying regions), it appears that generation of the full-length PCR product depends not only on the chemical structures of the substitution and the nature of the polymerase but also on whether the substitution is on dU or dC. Furthermore, the template sequence greatly affected generation of the PCR product, depending on the combination of the DNA polymerase and modified triphosphate. By examining primer extension reactions using primers and templates containing C5-modified dUs, we found that a modified dU at the 3' end of the elongation strand greatly affects the catalytic efficiency of DNA polymerases, whereas a modified dU opposite the elongation site on the template strand has less of an influence on the catalytic efficiency.

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Systematic characterization of 2′-deoxynucleoside- 5′-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA

Thymidine analogues bearing a variety of functional groups at the C5-position via an amino-linker arm were prepared and the substrate activity for PCR using thermophilic KOD Dash DNA polymerase was examined. The enzyme accepted the thymidine analogues bearing pyridine, imidazole, biotin, a cationic-charged guanidinium, a cationic-charged amino, mercaptopyridyl and phenanthrolne groups at the C5-position, forming the corresponding PCR product. However, a thymidine analogue bearing a carboxyl group at the C5-position was a poor substrate and the corresponding PCR products could not be obtained. The thymidine analogue bearing a mercapto group was also a poor substrate for the enzyme, because it dimerized by disulfide linkage under PCR conditions. The enzyme hardly accepts the thymidine analogues with a negatively-charged carboxyl group or a bulky group as a substrate. KOD Dash DNA polymerase, having a broader substrate specificity than any other DNA polymerase, will expand the variety of modified DNAs that can be prepared by PCR.

Expansion of repertoire of modified DNAs prepared by PCR using KOD Dash DNA polymerase.

The pyrimidine bases of RNA are uracil (U) and cytosine (C), while thymine (T) and C are used for DNA. The C(5) position of C and U is unsubstituted, whereas the C(5) of T is substituted with a Me group. Miller et al. hypothesized that various C(5)-substituted uracil derivatives were formed during chemical evolution, and that C(5)-substituted U derivatives may have played important roles in the transition from an 'RNA world' to a 'DNA-RNA-protein world'. Hyperthermophilic bacteria and archaea are considered to be primitive organisms that are evolutionarily close to the universal ancestor of all life on earth. Thus, we examined the substrate specificity of several C(5)-substituted or C(5)-unsubstituted dUTP and dCTP analogs for several DNA polymerases from hyperthermophilic bacteria, hyperthermophilic archaea, and viruses during PCR or primer extension reaction. The substrate specificity of the C(5)-substituted or C(5)-unsubstituted pyrimidine nucleotides varied greatly depending on the type of DNA polymerase. The significance of this difference in substrate specificity in terms of the origin and evolution of the DNA replication system is discussed briefly.

Differences in substrate specificity of C(5)-substituted or C(5)-unsubstituted pyrimidine nucleotides by DNA polymerases from thermophilic bacteria, archaea, and phages

A series of C5 substituted analogs of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate were synthesized and investigated their substrate properties for thermostable DNA polymerases during polymerase chain reaction (PCR). The PCR assays indicated that the relative yield of the full-length product is dependent on whether the substitution is on dU or dC, and on the nature of the polymerase.

Takehiro Tamura

Papers

Systematic characterization of 2′-deoxynucleoside- 5′-triphosphate analogs as substrates for DNA polymerases by polymerase chain reaction and kinetic studies on enzymatic production of modified DNA

Expansion of repertoire of modified DNAs prepared by PCR using KOD Dash DNA polymerase.

Differences in substrate specificity of C(5)-substituted or C(5)-unsubstituted pyrimidine nucleotides by DNA polymerases from thermophilic bacteria, archaea, and phages

Comparison study on PCR amplification of modified DNA by using various kinds of polymerase and modified nucleoside triphosphates.