Probing the function of nucleotides in the catalytic cores of the 8-17 and 10-23 DNAzymes by abasic nucleotide and C3 spacer substitutions.

8-17 and 10-23 are the two most comprehensively studied RNA-cleaving DNAzymes to date and have the ability to carry out sequence-specific cleavage of both all-RNA or chimeric RNA/DNA substrates. Mutagenesis studies of 8-17 and 10-23 DNAzymes using alternative natural nucleotides to substitute a given nucleotide in the DNAzyme sequence have found that both DNAzymes are able to tolerate a variety of alterations at many sequence locations. Chemical modification studies employing nucleotides containing nonnatural nucleobases have led to findings that some specific entities of selected nucleobases are irreplaceable by other functional groups.

Enzymatic cleavage of nucleic acids on gold nanoparticles: a generic platform for facile colorimetric biosensors.

The enzymatic cleavage of nucleic acids (DNA or DNA with a single RNA linkage) on well-dispersed gold nanoparticles (AuNPs) is exploited in the design of facile colorimetric biosensors. The assays are performed at salt concentrations such that DNA-modified AuNPs are barely stabilized by the electrostatic and steric stabilization. Enzymatic cleavage of DNA chains on the AuNP surface destabilizes the AuNPs, resulting in a rapid aggregation driven by van der Waals attraction, and a red-to-purple color change.

DNA aptamer folding on gold nanoparticles: from colloid chemistry to biosensors.

We have investigated the effect of the folding of DNA aptamers on the colloidal stability of gold nanoparticles (AuNPs) to which an aptamer is tethered. On the basis of the studies of two different aptamers (adenosine aptamer and K+ aptamer), we discovered a unique colloidal stabilization effect associated with aptamer folding: AuNPs to which folded aptamer structures are attached are more stable toward salt-induced aggregation than those tethered to unfolded aptamers. This colloidal stabilization effect is more significant when a DNA spacer was incorporated between AuNP and the aptamer or when lower aptamer surface graft densities were used.

Simple fluorescent sensors engineered with catalytic DNA 'MgZ' based on a non-classic allosteric design.

Most NAE (nucleic acid enzyme) sensors are composed of an RNA-cleaving catalytic motif and an aptameric receptor. They operate by activating or repressing the catalytic activity of a relevant NAE through the conformational change in the aptamer upon target binding. To transduce a molecular recognition event to a fluorescence signal, a fluorophore-quencher pair is attached to opposite ends of the RNA substrate such that when the NAE cleaves the substrate, an increased level of fluorescence can be generated.

Simple and rapid colorimetric enzyme sensing assays using non-crosslinking gold nanoparticle aggregation.

Non-crosslinking gold nanoparticle (AuNP) aggregation induced by the loss (or screen) of surface charges is applied for enzymatic activity sensing and potentially inhibitor screening.

Entrapment of fluorescence signaling DNA enzymes in sol-gel-derived materials for metal ion sensing.

Three fluorescence signaling DNA enzymes (deoxyribozymes or DNAzymes) were successfully immobilized within a series of sol-gel-derived matrixes and used for sensing of various metal ions. The DNAzymes are designed such that binding of appropriate metal ions induces the formation of a catalytic site that cleaves a ribonucleotide linkage within a DNA substrate. A fluorophore (fluorescein) and a quencher (DABCYL, [4-(4-dimethylaminophenylazo)benzoic acid]) were placed on the two deoxythymidines flanking the ribonucleotide to allow the generation of fluorescence upon the catalytic cleavage at the RNA linkage.

Efficient signaling platforms built from a small catalytic DNA and doubly labeled fluorogenic substrates.

RNA-cleaving deoxyribozyme 8-17 has been increasingly used in nanotechnology and biosensing applications. Conventional methods to equip 8-17 with fluorescent signaling property usually involve covalent attachment of two dyes at nucleotide positions that are far away from the catalytic core, such that the bulky dye structures would not affect the deoxyribozyme activity. However, the maximum fluorescent enhancement associated with these 8-17 constructs is typically < or =10-fold, due to a high fluorescent background.

Evolution of high-branching deoxyribozymes from a catalytic DNA with a three-way junction.

Here, we report the evolution of two star-shaped (five-way junction) deoxyribozymes from a catalytic DNA containing a three-way junction scaffold. The transition was shown to be a switch rather than a gradual progression. The star-shaped motifs, surprisingly, only took five selection cycles to be detected, and another four to dominate the evolving population.

Phosphoester-transfer mechanism of an RNA-cleaving acidic deoxyribozyme revealed by radioactivity tracking and enzymatic digestion.

A convenient method involving 32P-labeling of an RNA substrate at the cleavage site and subsequent enzymatic digestion of cleavage products via phosphatases reveals that pH4DZ1--an RNA-cleaving deoxyribozyme with optimal activity at pH 4--forms a 5'-cleavage fragment with 2',3'-cyclic phosphate group and a 3'-cleavage fragment with 5'-OH group.

Revitalization of six abandoned catalytic DNA species reveals a common three-way junction framework and diverse catalytic cores.

A library containing as many as 10(16) nucleic acid candidates is typically used to isolate artificial ribozymes and deoxyribozymes (DNAzymes) in an in vitro selection experiment, with only a handful of sequences surviving many rounds of stringent selection steps. These winning species are generally the focus of interest whereas the less competitive contenders are usually not examined. Nevertheless, molecular species abandoned during the selection process might still represent a rich pool of catalytic motifs that are useful for the examination of DNA's inherent catalytic ability, and for the design of molecular tools for practical applications.

Quenching of fluorophore-labeled DNA oligonucleotides by divalent metal ions: implications for selection, design, and applications of signaling aptamers and signaling deoxyribozymes.

Recent years have seen a dramatic increase in the use of fluorescence-signaling DNA aptamers and deoxyribozymes as novel biosensing moieties. Many of these functional single-stranded DNA molecules are either engineered to function in the presence of divalent metal ion cofactors or designed as sensors for specific divalent metal ions. However, many divalent metal ions are potent fluorescence quenchers.

Making AppDNA using T4 DNA ligase.

5('),5(')-Adenylyl pyrophosphoryl DNA (AppDNA) contains a high-energy pyrophosphate linkage and can be exploited as an activated DNA substrate to derive new DNA enzymes for carrying out various DNA modification reactions. For this reason, enzymatic synthesis of AppDNA is highly desirable. AppDNA is a known intermediate in DNA ligase mediated DNA ligation reactions, but rarely accumulates under normal reaction conditions.