RNA, RNA interference, ribozyme switches, microRNA, structural studies, aptamers, and more ...
The importance of RNA interference was recognized by the Nobel Assembly, who awarded the 2006 Nobel Prize in Physiology or Medicine jointly to Andrew Z. Fire (Stanford School of Medicine, USA) and Craig C. Mello (University of Massachusetts, USA) for their discovery of RNA interference—gene silencing by double-stranded RNA.
Thomas Tuschl, winner of the Wiley Prize in Biomedical Sciences in 2003, discussed RNA Interference and Small Interfering RNAs in a Minireview in ChemBioChem, 2001, 2, 239.
The assembly of RNA oligomers relies principally on solid-phase approaches, although some alternative methods have been developed to date. A highly efficient and practical method of liquid-phase synthesis for RNA oligomers by using alkyl-chain-type soluble support is reported. The utility of the liquid-phase method through 21-mer RNA synthesis on a gram scale is described (see scheme).
Put a cap on it: Hairpin-shaped RNAs and dumbbell-shaped RNAs were prepared using a thiol–maleimino Michael addition and exhibited good serum and thermal stability. These capped structures were shown to be cleaved by Dicer and RNA interference (RNAi) experiments showed that RhpRNA (see picture, top right) was highly efficient at RNAi with an IC50 value of 6 pM.
Seeing is believing: Different miRNAs were used as templates for the photoreduction of an azide-based immolative linker by RuII-peptide nucleic acid (PNA) conjugates to uncage rhodamine (see figure). The method was validated by using two sets of γ-serine-modified PNA derivatives with varying affinities to their target (perfect match and mismatch).
Intrinsic pKa shifts between 0 and 0.8 pK units of the eight N sites in the single-stranded DNA hexanucleoside pentaphosphate d(ApGpGpCpCpT) were determined by NMR spectroscopic analysis and comparison with the pKa values of the individual nucleosides. Extensive neighboring effects are due to stacking and charge repulsion. The existing tautomeric equilibria are characterized in detail.
Know your limit! IsoGNA (a structural isomer of GNA) was found—in sharp contrast to GNA—to be highly restricted in its ability to base-pair with itself and other nucleic acids. While homogeneous sequences (e.g. isoGNA(A)16) formed duplexes, the heterogeneous sequences showed no base-pairing. This exemplifies the limitations of canonical nucleobases as the recognition elements in simpler, more primitive phosphate backbones.
A twist to DNA structure: Moving the phosphodiester backbone from the natural C(5') position into the C(6') position, which is only available on the bicyclo-DNA skeleton, leads to isobicyclo-DNA (Isobc-DNA; see scheme). Isobc-DNA forms stable duplexes with DNA and discriminates RNA as a complement. In addition, it forms very stable antiparallel self-duplexes with a structure that is different from A-, B-, and Z-DNA.
Detection with confidence: A biosensing strategy based on RNA structure-switching aptamers utilises an internal control to distinguish target binding from false-positive (chemical or nuclease degradation) signals. The strategy uses the exceptional affinity and specificity that numerous RNA aptamers exhibit as molecular recognition elements.
Suppressing the urge to translate: Sequences with the potential to form G-quadruplexes were identified in the open reading frames of E. coli genes. These sequences were found to form parallel G-quadruplexes and to suppress translation (see picture) of the mRNAs into proteins both in vitro and in cells.
Dynamics of initiation: A stepwise reaction of translation initiation can be evaluated by using a quartz crystal microbalance (QCM) in real time (see figure). This system permitted the direct observation of the dynamics of initiation factors (IF) on ribosomes during initiation complex (IC) formation. The results suggest that IF3 binds and stays bound to ICs, and its interaction mode is altered during the formation of 30S-IC and 70S-IC and is finally induced to dissociate from ICs by 50S binding.
Writer's blocks: The first synthesis of RNA purine building blocks, 4'-selenoadenosine and 4'-selenoguanosine was achieved from D-ribose by regioisomeric rearrangement, which was confirmed by X-ray crystallography. 4'-Selenoadenosine exists in an unusual mixture of north and south conformers in the solid state.
Well red: A protein-RNA crosslinker has been genetically encoded that can be controlled with red light (see scheme), thus offering high penetration depths in biological materials. This should enable the discovery and mapping of transient protein–RNA interactions and enable the design of peptide- and protein-based drugs for RNA-targeted photodynamic therapy.
Green for GO: A label-free approach with multiple enhancement of the signal for the determination of microRNA has been developed by taking advantage of a novel graphene oxide (GO)/intercalating dye based fluorescent hairpin probe and an isothermal polymerization reaction (see scheme; SYBR Green 1: reporter molecule).
Discrimination in the workplace: A peptide nucleic acid (PNA)-derived probe, in which PNA forms a pseudocomplementary heteroduplex with inosine-containing DNA or RNA, effectively discriminates a single-nucleotide difference in a closely related group of sequences of single-stranded DNA and/or RNA (see figure).
Novel 2'-RNA protection: The reaction of 2-cyano-2-methyl propanal with 2'-O-aminooxymethylribonucleosides leads to 2'-O-(2-cyano-2,2-dimethylethanimine-N-oxymethyl)ribonucleosides, which were then converted into their fully protected phosphoramidite derivatives (see scheme; DMTr=4,4'-dimethoxytrityl). These amidites were successfully employed in the solid-phase synthesis of three chimeric RNA sequences, each with a different purine/pyrimidine ratio. This approach is straightforward, efficient, and cost effective.
Complex to clear: An outwardly complex mixture, arising from mixing glycolaldehyde and cyanamide, was analysed by pure-shift DOSY NMR spectroscopy (see scheme). Amongst the species identified were potential threose nucleic acid (TNA) and RNA nucleoside precursors. These observations may have implications for the potentially prebiotic assembly chemistry of pyrimidine threonucleotides, and therefore of TNA.
Chemical plant: An efficient synthesis of the hypermodified natural tRNA modifications wybutosine (yW) and hydroxywybutosine (OHyW) is reported (see scheme). The preparation of isotope-labeled analogues of yW and OHyW is described, which are used for precise quantification of yW and OHyW in the plant species Arabidopsis thaliana.
N-genering oligos: A new class of nucleotide scaffold based on N-ethyl-N-coupled nucleosides (BC/A) has been designed. MD simulations of 3'-BC-DNA: Klenow fragment complexes show disruption of the 3'-exonuclease active site. This modification confers extraordinary resistance against 3'-exonucleases and is accepted by the RNAi machinery.
Both protonated and deuterated samples were employed in the study of the L7Ae box C/D RNA complex by 1H-detected solid-state NMR spectroscopy. This approach yielded high-resolution spectra and was used to determine the intermolecular interface and extract structural parameters with high accuracy.
The complex class of cobalamin-sensitive riboswitches use a “kissing loop” to regulate gene expression. The molecular details of the recognition and folding mode of this riboswitch have been revealed by recent X-ray structures. These insights, together with the identification of RNA polymerase pause sites during transcription, have resulted in a more complete understanding of the response mechanism. AdoCbl=adenosylcobalamin.
Lighting the way with DNA: Molecular beacon micelle flares (MBMFs), based on self-assembly of diacyllipid–molecular-beacon conjugates (L-MBs; see figure), have been developed for combined mRNA detection and gene therapy. These MBMFs were shown to inhibit a model gene in vitro and decrease the viability of cancer cells in culture.
Killing the message: An approach to direct the cleavage of RNA targets with small molecules in living cells is described (see scheme). A bifunctional small molecule (purple) that recognizes a specific three nucleotide repeat sequence and cleaves that sequence in response to light was shown to be effective at degrading the myotonic dystrophy type 1 (DM1) extended repeat RNAs, thereby affecting biological functions.
Keeping a broad (RNA) perspective: Conventional biochemical detection systems only have a 100-fold linear response range. The range of potassium concentrations detected by an RNA G-quadruplex sequence can be broadened by intentionally populating multiple intermediate folding states (see scheme). The folding of the RNA G-quadruplexes was monitored by both circular dichroism and intrinsic fluorescence spectroscopy.
RNA, just another starting material? Nobel Laureate Tom Cech shows that with an education steeped in kinetics, thermodynamics, and molecular structure, and armed with the ability to synthesize molecules, the chemist is ideally suited to investigate RNA.
On target: We have developed two cascade amplification strategies that combine duplex specific nuclease (DSN) amplicon with either G-quadruplex-based DNA peroxidase or 8–17 DNAzyme amplicon for miRNA detection. In this way, sensitive and convenient detection of miRNAs was achieved. In the DNA peroxidase-based system, a visual color change could be observed in the presence of target miRNAs (see scheme).
Fluorishing: The Togni reagent allows efficient synthetic access to fluorine-labeled RNA molecules (see picture). These are in turn highly useful for NMR spectroscopic analyses of secondary and tertiary structures, RNA–protein interactions, and functionality of riboswitch modules.
Loop conformation: The loop of the RNA domain helix 69 (H69) was modified with the fluorescent analogue 2-aminopurine (2AP), thus showing different conformational states under various conditions. The application of this model RNA reveals the unique impact of aminoglycoside neomycin, which differs from the effects of structurally related compounds paromomycin and gentamicin, on the H69 loop conformation in solution (see picture).
It all clicks into place: A potent telomere-targeting small molecule has been identified by using the copper-free 1,3-dipolar cycloaddition of a series of alkyne and azide building blocks catalyzed by a non-Watson–Crick DNA secondary structure (see picture). This method rapidly identifies, otherwise unanticipated, potent small-molecule probes to selectively target a given RNA or DNA.
Checking for mistakes: By conjugating a catalytic domain with a guide RNA, deamination activity can be harnessed to repair a specific codon on mRNA. This method can be used for the highly selective repair of point mutations in mRNA by site-selective editing.
Useful diversity: Quantification of modified tRNA nucleobases in different murine and porcine tissues reveals a tissue-specific overall modification content. The modification content correlates with rates of protein synthesis in vitro, suggesting a direct link between tRNA modification levels and tissue-specific translational efficiency.
Besides their four canonical nucleobases, DNA and RNA contain a variety of highly modified nucleosides including the epigenetic bases mC, hmC, fC, and caC (see scheme), which are able to increase the chemical information content. The function of these modified bases is to generate a second level of chemical complexity in RNA and DNA in addition to the first coding level provided by the sequence of the canonical Watson–Crick bases.
Fingering RNA: To study the function of the variety of RNA species that have been discovered recently, sequence-specific RNA-binding molecules with tunable specificity are required. Here, the use of zinc fingers (ZFs) from the splicing factors ZRANB2 and RBM5 as potential modules for the assembly of sequence-specific RNA-binding molecules is demonstrated.
An ancient reaction vessel: TobZ carbamoylates the antibiotic tobramycin to form nebramycin 5'. The YrdC-like domain (blue) catalyzes the formation of the novel intermediate carbamoyladenylate, which is channeled through a common “reaction chamber” to the Kae1-like domain (brown), site of carbamoyl transfer.
The application of EPR spectroscopy to study of the structures and dynamics of nucleic acids requires site-specific incorporation of stable free radicals (spin labels), known as site-directed spin labelling (SDSL). This review recapitulates the three main strategies used for SDSL of nucleic acids: spin labelling during oligonucleotide synthesis, post-synthetic labelling and noncovalent labelling.