DNA (and RNA) molecules can as directed by their sequence fold into a variety of two and three dimensional structures. Many are found in nature, most prominently exemplified by structured RNA molecules such as tRNA, ribosomal RNA, ribozymes and riboswitches, but more complex DNA structures such as triplexes, quadruplexes and three and four way junctions are also found. However, within the past couple of decades an increasing number of artificial DNA and RNA structures have been discovered and developed within DNA nanotechnology.
Prominent current ideas on how life emerged on Earth include an RNA world hypothesis in which RNA performed informational as well as catalytic functions in the absence of both DNA and protein. Demonstration of a self-replicative system based on ribonucleic acid polymers as both information carriers and catalysts would lend support to such a scenario. A pivotal component of this system would be an RNA dependent RNA polymerase ribozyme capable of replicating its own RNA gene. Recent work from the Holliger group at the Laboratory for Molecular Biology in Cambridge has provided synthetic ribozymes1 that just might foreshadow the future engineering of such self-replicative systems.
Noncovalent binding and fluorogenic response of cyanine dyes to DNA homoquadruplex and PNA-DNA heteroquadruplex structures
Two symmetrical cyanine dyes based on benzothiazole heterocycles and a trimethine bridge were found to bind to a parallel-stranded DNA guanine quadruplex based on the MYC oncogene promoter sequence with high nanomolar affinity and 1:1 stoichiometry. The dyes exhibited substantial fluorescence enhancements upon binding. In the presence of homologous guanine-rich peptide nucleic acid oligomers, PNA-DNA heteroquadruplexes were formed. The dyes retained their ability to bind to the heteroquadruplexes at low micromolar concentrations and with varying fluorescence enhancements, although indeterminate stoichiometries preclude quantitative comparison of the affinities with the DNA homoquadruplex precursor. The difference in fluorescence enhancement between DNA homoquadruplex and PNA-DNA heteroquadruplex allows the dyes to be used as fluorogenic indicators of hybridization in a facile method for determining PNA-DNA stoichiometry.
Pyrrolidinyl peptide nucleic acid with α/β-peptide backbone: A conformationally constrained PNA with unusual hybridization properties
We describe herein a new conformationally constrained analog of PNA carrying an alternating α/β amino acid backbone consisting of (2'R,4'R)-nucleobase-subtituted proline and (1S,2S)-2-aminocyclopentanecarboxylic acid (acpcPNA). The acpcPNA has been synthesized and evaluated for DNA, RNA and self-pairing properties by thermal denaturation experiments. They are shown to form antiparallel hybrids with complementary DNA with high affinity and sequence specificity. Unlike other PNA systems, the thermal stability of acpcPNA·DNA hybrid is largely independent of G+C contents, and is generally higher than that of acpcPNA·RNA hybrid with the same sequence. Thermodynamic parameters analysis suggests that the A·T base pairs in the acpcPNA·DNA hybrids is enthalpically stabilized over G·C pairs. The acpcPNA also shows a hitherto unreported behavior, namely the inability to form self-pairing hybrids. These unusual properties should make the new acpcPNA a potentially useful candidate for various applications including microarray probes and antigene agents.
Sensitive detection of nucleic acids by PNA hybridization directed co-localization of fluorescent beads
We have designed a pair of biotinylated peptide nucleic acid (PNA) probes targeting two sequences in 18S rRNA (from the parasite Trypanosoma brucei) at a distance of 191 bp (ca. 60 nm) from each other. The PNA probes were individually bound to (strept)avidin coated fluorescent beads, differing in size and color (green beads (1 µm) and red beads (5.9 µm)), thereby allowing distinct detection of each PNA probe by conventional fluorescence microscopy. These two PNA beads showed easily detectable co-localization when simultaneously hybridizing to a target nucleic acid. The assay detected the parasite 18S rRNA down to 1.6 fmol while there was no such co-localization visible with human 18S rRNA not containing the PNA targets. Furthermore, the assay showed positive detection with 1.6 ng of total RNA (correspond to RNA from ca. 300 parasites). Upon further optimization this method may provide a new tool for a diagnosis of HAT and may more generally have applications within diagnostics for (neglected) infectious diseases.