RNA Loop Research Articles

Thermodynamic Contributions of Divalent Ion Interactions with a Symmetric Internal Loop in RNA

Folding of single stranded RNA upon itself generates complex structures containing many bulges and loops along with A‐form helical regions. These structures contain many structural motifs that are expected to be sites for inter‐ and intra‐molecular interactions. Motifs in which phosphate bonds are in close proximity are expected to require interactions with metal ions to stabilize the RNA structure Thermodynamic studies on an asymmetrical trinucleotide bulge RNA in our laboratory have shown that the divalent ions stabilize the RNA by 3‐5 kcal/mol ( ? ?G° relative to zero). In this study we are examining the thermodynamic properties of symmetrical internal loops and their interactions with divalent ion. We are examining a 2x2 symmetric bulge derived from magnesium binding region of the thiamine pyrophosphate. Initial thermodynamic analysis shows that the symmetrical loop interacts with magnesium ions and is stabilized approximately 1.5 kcal/mol relative to the 0mM buffer. The helix without the bulge was stabilized approximately 2 kcal/mol relative to the 0mM buffer. The competition between monovalent and divalent ions in RNA interactions is also being examined. This work was funded by NSF MCB‐0621509 to NG.

Evidence of RNA looping by PTB using Fluorescence Resonance Energy Transfer and NMR spectroscopy

In eukaryotes, alternative splicing plays an important role in proteome diversity and sound protein expression. Polypyrimidine tract binding protein (PTB) is a major alternative splicing factor that has been involved mostly with splicing repression. It has been proposed that the mechanism of action of PTB involves a looping out of exons flanked by pyrimidine tracts. Here, we present Fluorescence, NMR and single molecule data that directly support this mechanism. We show that the RNA binding domains 3 and 4 of PTB (RBD34) bind two distant pyrimidine‐tracts and can bring their 5' and 3' ends in close proximity, thus looping the RNA. Looping efficiency depends on the length of the intervening sequence with preference for a 15‐nucleotide spacer between the pyrimidine‐tracts. NMR chemical shifts perturbation show a specific directionality of the PTB inter‐action with RNA, RRM3 and RRM4 binding the 5' and the 3' pyrimidine‐tract, respectively. A PTB mutant reveals a synergistic effect between RBD3 and 4 for efficient looping in vitro and repress splicing in vivo.

Crystal Structure of an RluF–RNA Complex: A Base-Pair Rearrangement Is the Key to Selectivity of RluF for U2604 of the Ribosome

Escherichia coli pseudouridine synthase RluF is dedicated to modifying U2604 in a stem–loop of 23S RNA, while a homologue, RluB, modifies the adjacent base, U2605. Both uridines are in the same RNA stem, separated by ∼ 4 Å. The 3.0 Å X-ray crystal structure of RluF bound to the isolated stem–loop, in which U2604 is substituted by 5-fluorouridine to prevent catalytic turnover, shows RluF distinguishes closely spaced bases in similar environments by a selectivity mechanism based on a frameshift in base pairing. The RNA stem–loop is bound to a conserved binding groove in the catalytic domain. A base from a bulge in the stem, A2602, has folded into the stem, forcing one strand of the RNA stem to translate by one position and thus positioning U2604 to flip into the active site. RluF does not modify U2604 in mutant stem–loops that lack the A2602 bulge and shows dramatically higher activity for a stem–loop with a mutation designed to facilitate A2602 refolding into the stem with concomitant RNA strand translation. Residues whose side chains contact rearranged bases in the bound stem–loop, while conserved among RluFs, are not conserved between RluFs and RluBs, suggesting that RluB does not bind to the rearranged stem loop.

Characterization of pokeweed antiviral protein binding to mRNA cap analogs: Competition with nucleotides and enhancement by translation initiation factor iso4G

Pokeweed antiviral protein (PAP) is a type I ribosomal inactivating protein (RIP). PAP binds to and depurinates the sarcin/ricin loop (SRL) of ribosomal RNA resulting in the cessation of protein synthesis. PAP has also been shown to bind to mRNA cap analogs and depurinate mRNA downstream of the cap structure. The biological role of cap binding and its possible role in PAP activity are not known. Here we show the first direct quantitative evidence for PAP binding to the cap analog m(7)GTP. We report a binding affinity of 43.3+/-0.1 nM at 25 degrees C as determined by fluorescence quenching experiments. This is similar to the values reported for wheat cap-binding proteins eIFiso4E and eIFiso4F. van't Hoff analysis of m(7)GTP-PAP equilibrium reveals a binding reaction that is enthalpy driven and entropy favored with TDeltaS degrees contributing 15% to the overall value of DeltaG degrees . This is in contrast to the wheat cap-binding proteins which are enthalpically driven in the DeltaG degrees for binding. Competition experiments indicate that ATP and GTP compete for the cap-binding site on PAP with slightly different affinities. Fluorescence studies of PAP-eIFiso4G binding reveal a protein-protein interaction with a K(d) of 108.4+/-0.3 nM. eIFiso4G was shown to enhance the interaction of PAP with m(7)GTP cap analog by 2.4-fold. These results suggest the involvement of PAP-translation initiation factor complexes in RNA selection and depurination.

P21 Ras/Impedes Mitogenic Signal Propagation Regulates Cytokine Production and Migration in CD4 T Cells

The propensity of T cells to generate coordinated cytokine responses is critical for the host to develop resistance to pathogens while maintaining the state of immunotolerance to self-antigens. The exact mechanisms responsible for preventing the overproduction of proinflammatory cytokines including interferon (IFN)-gamma are not fully understood, however. In this study, we examined the role of a recently described Ras GTPase effector and repressor of the Raf/MEK/ERK cascade called impedes mitogenic signal propagation (Imp) in limiting the induction of T-cell cytokines. We found that stimulation of the T cell receptor complex leads to the rapid development of a physical association between Ras and Imp. Consistent with the hypothesis that Imp inhibits signal transduction, we also found that disengagement of this molecule by the Ras(V12G37) effector loop mutant or RNA interference markedly enhances the activation of the NFAT transcription factor and IFN-gamma secretion. A strong output of IFN-gamma is responsible for the distinct lymphocyte traffic pattern observed in vivo because the transgenic or retroviral expression of Ras(V12G37) caused T cells to accumulate preferentially in the lymph nodes and delayed their escape from the lymphoid tissue, respectively. Together, our results describe a hitherto unrecognized negative regulatory role for Imp in the production of IFN-gamma in T cells and point to Ras-Imp binding as an attractive target for therapeutic interventions in conditions involving the production of this inflammatory cytokine.

Small hairpin loop RNA targeting HIF-1α down-regulates VEGF and up-regulates PEDF in human retinal pigment epithelial cells under hypoxic condition

The aim of this study was to explore the effect of small hairpin loop RNA (shRNA) silencing hypoxia-induced factor 1alpha (HIF-1alpha) gene on the expression of vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) in human retinal pigment epithelium (RPE) cells under hypoxic condition. Two target sites of HIF-1alpha mRNA were chosen and two kinds of shRNA were designed and synthesized against the target sites. Then the two kinds of shRNA were transfected into human RPE cells in vitro, respectively. These cells were cultured under hypoxic condition that was simulated by using 150 mumol/L CoCl(2). The mRNA expressions of HIF-1alpha, VEGF and PEDF were tested by semi-quantitative reverse transcription PCR (RT-PCR). The protein levels of HIF-1alpha, VEGF and PEDF were analyzed by Western blotting. After the two kinds of HIF-1alpha-specific shRNA were transfected into RPE cells respectively, the expression of HIF-1alpha mRNA and the levels of HIF-1alpha protein were decreased significantly in RPE cells under hypoxic condition. The expression of VEGF mRNA and the levels of protein significantly were also decreased. However, the levels of PEDF protein was significantly increased, but the expression of PEDF mRNA showed no significant changes. In conclusion, HIF-1alpha-specific shRNA can effectively silence the HIF-1alpha gene, and consequently down-regulate VEGF and up-regulate PEDF expression against hypoxia. These results reveal that HIF-1 is associated with posttranslational mechanism for down-regulating PEDF under hypoxia and provide an explanation for hypoxia-provoked increases in VEGF/PEDF ratios. These results also suggest that HIF-1 is one of the key cytokines to retinal neovascularization.

Two-Dimensional Combinatorial Screening Identifies Specific Aminoglycoside−RNA Internal Loop Partners

Herein is described the identification of RNA internal loops that bind to derivatives of neomycin B, neamine, tobramycin, and kanamycin A. RNA loop-ligand partners were identified by a two-dimensional combinatorial screening (2DCS) platform that probes RNA and chemical spaces simultaneously. In 2DCS, an aminoglycoside library immobilized onto an agarose microarray was probed for binding to a 3 x 3 nucleotide RNA internal loop library (81,920 interactions probed in duplicate in a single experiment). RNAs that bound aminoglycosides were harvested from the array via gel excision. RNA internal loop preferences for three aminoglycosides were identified from statistical analysis of selected structures. This provides consensus RNA internal loops that bind these structures and include: loops with potential GA pairs for the neomycin derivative, loops with potential GG pairs for the tobramycin derivative, and pyrimidine-rich loops for the kanamycin A derivative. Results with the neamine derivative show that it binds a variety of loops, including loops that contain potential GA pairs that also recognize the neomycin B derivative. All studied selected internal loops are specific for the aminoglycoside that they were selected to bind. Specificity was quantified for 16 selected internal loops by studying their binding to each of the arrayed aminoglycosides. Specificities ranged from 2- to 80-fold with an average specificity of 20-fold. These studies show that 2DCS is a unique platform to probe RNA and chemical space simultaneously to identify specific RNA motif-ligand interactions.

Liquid-crystal NMR structure of HIV TAR RNA bound to its SELEX RNA aptamer reveals the origins of the high stability of the complex

Transactivation-response element (TAR) is a stable stem-loop structure of HIV RNA, which plays a crucial role during the life cycle of the virus. The apical loop of TAR acts as a binding site for essential cellular cofactors required for the replication of HIV. High-affinity aptamers directed against the apical loop of TAR have been identified by the SELEX approach. The RNA aptamers with the highest affinity for TAR fold as hairpins and form kissing complexes with the targeted RNA through loop-loop interactions. The aptamers with the strongest binding properties all possess a GA base pair combination at the loop-closing position. Using liquid-crystal NMR methodology, we have obtained a structural model in solution of a TAR-aptamer kissing complex with an unprecedented accuracy. This high-resolution structure reveals that the GA base pair is unilaterally shifted toward the 5' strand and is stabilized by a network of intersugar hydrogen bonds. This specific conformation of the GA base pair allows for the formation of two supplementary stable base-pair interactions. By systematic permutations of the loop-closing base pair, we establish that the identified atomic interactions, which form the basis for the high stability of the complex, are maintained in several other kissing complexes. This study rationalizes the stabilizing role of the loop-closing GA base pairs in kissing complexes and may help the development or improvement of drugs against RNA loops of viruses or pathogens as well as the conception of biochemical tools targeting RNA hairpins involved in important biological functions.

Conserved unpaired adenine residues are important for ordered structures of 5S ribosomal RNA. An infrared study of the secondary and tertiary structure of Thermus thermophilus 5S rRNA.

An improved set of infrared calibration spectra for the determination of G X C and A X U base pairs leads to 32 +/- 3 G X C (+ G X U) and 4 +/- 1 A X U base pairs for Thermus thermophilus 5S RNA in the presence and absence of Mg2+. These results give further support for the consensus secondary structure of 5S RNA recently proposed by several groups. T. thermophilus 5S RNA shows, in the presence of Mg2+, a distinct two-step thermal melting of its ordered structure. Based on new data about the stacking dependence of infrared intensities of unpaired ribonucleotides the spectral changes of the low-temperature transition should be explained by melting of stacked arrangements of unpaired bases and/or non-standard base pairs. Striking is the reduction in A stacking, which is not related to the melting of A X U base pairs, indicating the importance of the mostly conserved unpaired adenines for the Mg2+ stabilized higher-order structures especially within internal loops of 5S RNA.

2-Aminopurine Fluorescence: Discrimination Between Specific and Unspecific Ligand Binding to the Kissing-Loop Dimer of the HIV-1 RNA

The fluorescent 2-aminopurine probe (2-AP) incorporated into the loop of 23-mer RNA hairpin of HIV-1 genome dimerization initiation site (DIS) was used for discrimination of specific and unspecific binding of paromomycin and spermine to the kissing loop dimer (KD) formed in solution. While both ligands stabilized the KD RNA structure, only paromomycin binding resulted in significant increase of 2-AP fluorescence. These observations suggest that the 2-AP fluorescent RNA construct might be useful for selecting ligands specifically binding the HIV-1 kissing loop RNA dimer.

The Antitermin ation Activity of Bacteriophage λ N Protein is Controlled by the Kinetics of an RNA-Looping-Facilitated Interaction with the Transcription Complex

Protein N of bacteriophage λ activates the lytic phase of phage development in infected Escherichia coli cells by suppressing the activity of transcriptional terminators that prevent the synthesis of essential phage proteins. N binds tightly to the boxB RNA hairpin located near the 5′ end of the nascent pL and pR transcripts and induces an antitermination response in the RNA polymerase (RNAP) of elongation complexes located at terminators far downstream. Here we test an RNA looping model for this N-dependent “action at a distance” by cleaving the nascent transcript between boxB and RNAP during transcript elongation. Cleavage decreases antitermination, showing that an intact RNA transcript is required to stabilize the interaction of boxB-bound N with RNAP during transcription. In contrast, an antitermination complex that also contains Nus factors retains N-dependent activity after transcript cleavage, suggesting that these host factors further stabilize the N–RNAP interaction. Thus, the binding of N alone to RNAP is controlled by an RNA looping equilibrium, but after formation of the initial RNA loop and in the presence of Nus factors the system no longer equilibrates on the transcription time scale, meaning that the “range” of antitermination activity along the template in the full antitermination system is kinetically controlled by the dissociation rate of the stabilized N–RNAP complex. Theoretical calculations of nucleic acid end-to-end contact probabilities are used to estimate the local concentrations of boxB-bound N at elongation complexes poised at terminators, and are combined with N activity measurements at various boxB-to-terminator distances to obtain an intrinsic affinity (Kd) of ∼2×10−5 M for the N–RNAP interaction. This RNA looping approach is extended to include the effects of N binding at nonspecific RNA sites on the transcript and the implications for transcription control in other regulatory systems are discussed.

Structure of a TrmA–RNA complex: A consensus RNA fold contributes to substrate selectivity and catalysis in m 5 U methyltransferases

TrmA catalyzes S-adenosylmethionine (AdoMet)-dependent methylation of U54 in most tRNAs. We solved the structure of the Escherichia coli 5-methyluridine (m(5)U) 54 tRNA methyltransferase (MTase) TrmA in a covalent complex with a 19-nt T arm analog to 2.4-A resolution. Mutation of the TrmA catalytic base Glu-358 to Gln arrested catalysis and allowed isolation of the covalent TrmA-RNA complex for crystallization. The protein-RNA interface includes 6 nt of the T loop and two proximal base pairs of the stem. U54 is flipped out of the loop into the active site. A58 occupies the space of the everted U54 and is part of a collinear base stack G53-A58-G57-C56-U55. The RNA fold is different from T loop conformations in unbound tRNA or T arm analogs, but nearly identical to the fold of the RNA loop bound at the active site of the m(5)U MTase RumA. In both enzymes, this consensus fold presents the target U and the following two bases to a conserved binding groove on the protein. Outside of this fold, the RumA and TrmA substrates have completely different structures and protein interfaces. Loop residues other than the target U54 make more than half of their hydrogen bonds to the protein via sugar-phosphate moieties, accounting, in part, for the broad consensus sequence for TrmA substrates.

ZAK: a MAP3Kinase that transduces Shiga toxin- and ricin-induced proinflammatory cytokine expression

Shiga toxins (Stxs) and ricin initiate damage to host cells by cleaving a single adenine residue on the alpha-sarcin loop of the 28S ribosomal RNA. This molecular insult results in a cascade of intracellular events termed the ribotoxic stress response (RSR). Although Stxs and ricin have been shown to cause the RSR, the mitogen-activated protein kinase kinase kinase (MAP3K) that transduces the signal from intoxicated ribosomes to activate SAPKinases has remained elusive. We show in vitro that DHP-2 (7-[3-fluoro-4-aminophenyl-(4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl))]-quinoline), a zipper sterile-alpha-motif kinase (ZAK)-specific inhibitor, blocks Stx2/ricin-induced SAPKinase activation. Treatment of cells with DHP-2 also blocks Stx2/ricin-mediated upregulation of the proinflammatory cytokine interleukin-8 and results in a modest but statistically significant improvement in cell viability following Stx2/ricin treatment. Finally we show that siRNA directed against the N-terminus of ZAK diminishes Stx2/Ricin-induced SAPKinase activation. Together, these data demonstrate that a ZAK isoform(s) is the MAP3Kinase that transduces the RSR. Therefore, ZAKalpha and/or beta isoforms may act as potential therapeutic target(s) for treating Stx/ricin-associated illnesses. Furthermore, a small molecule inhibitor like DHP-2 may prove valuable in preventing the Stx/ricin-induced proinflammatory and/or apoptotic effects that are thought to contribute to pathogenesis by Stx-producing Escherichia coli and ricin.

Loop dependence of the stability and dynamics of nucleic acid hairpins

Hairpin loops are critical to the formation of nucleic acid secondary structure, and to their function. Previous studies revealed a steep dependence of single-stranded DNA (ssDNA) hairpin stability with length of the loop (L) as ∼L8.5 ± 0.5, in 100 mM NaCl, which was attributed to intraloop stacking interactions. In this article, the loop-size dependence of RNA hairpin stabilities and their folding/unfolding kinetics were monitored with laser temperature-jump spectroscopy. Our results suggest that similar mechanisms stabilize small ssDNA and RNA loops, and show that salt contributes significantly to the dependence of hairpin stability on loop size. In 2.5 mM MgCl2, the stabilities of both ssDNA and RNA hairpins scale as ∼L4 ± 0.5, indicating that the intraloop interactions are weaker in the presence of Mg2+. Interestingly, the folding times for ssDNA hairpins (in 100 mM NaCl) and RNA hairpins (in 2.5 mM MgCl2) are similar despite differences in the salt conditions and the stem sequence, and increase similarly with loop size, ∼L2.2 ± 0.5 and ∼L2.6 ± 0.5, respectively. These results suggest that hairpins with small loops may be specifically stabilized by interactions of the Na+ ions with the loops. The results also reinforce the idea that folding times are dominated by an entropic search for the correct nucleating conformation.

Down-regulation of vascular endothelial growth factor in human retinal pigment epithelial cells by small hairpin loop RNA targeting hypoxia inducible factor-1 alpha under hypoxia condition

To explore the effect of hypoxia inducible factor-1 alpha (HIF-1 alpha) gene on the expression of vascular endothelial growth factor (VEGF) in human retinal pigment epithelial (hRPE) cells under hypoxia conditions by using small hairpin loop RNA (shRNA) to silence HIF-1 alpha. CoCl(2) (150 micromol/L) was used to simulate the hypoxia environment for hRPE cells. After choosing a target site of HIF-1 alpha mRNA, shRNA was designed and synthesized by this target site. hRPE cells were transfected by this shRNA in vitro. Then, these cells were cultured under hypoxia conditions (150 micromol/L CoCl(2)). The mRNA expression of HIF-1 alpha and VEGF was measured by semi-quantitative reverse transcription PCR (RT-PCR). The protein level of HIF-1 alpha and VEGF was studied by western blot analysis. After hRPE cells were transfected by HIF-1 alpha-specific shRNA, RT-PCR showed that the expression of HIF-1 alpha mRNA was inhibited by 77.1%, and western blot analysis showed that the level of HIF-1 alpha protein was significantly decreased in hRPE cells under hypoxia conditions. Moreover, the expression of VEGF mRNA was inhibited by 27.8% and the level of VEGF protein was also significantly decreased in transfected hRPE cells under hypoxia conditions. Under hypoxia conditions, HIF-1 alpha-specific shRNA effectively keeps HIF-1 alpha gene silenced, and consequently down-regulates VEGF expression against hypoxia. These results suggest that HIF-1 alpha is one of the most important cytokines for retinal neovascularization.

P68 RNA Helicase Unwinds the Human let-7 MicroRNA Precursor Duplex and Is Required for let-7-directed Silencing of Gene Expression

MicroRNAs are short, single-stranded RNAs that arise from a transient precursor duplex. We have identified a novel activity in HeLa cell extracts that can unwind the let-7 microRNA duplex. Using partially purified material, we have shown that microRNA helicase activity requires ATP and has a native molecular mass of approximately 68 kDa. Affinity purification of the unwinding activity revealed co-purification of P68 RNA helicase. Importantly, recombinant P68 RNA helicase was sufficient to unwind the let-7 duplex. Moreover, like its native homolog, P68 RNA helicase did not unwind an analogous small interfering RNA duplex. We further showed that knockdown of P68 inhibited let-7 microRNA function. From our data, we conclude that P68 RNA helicase is an essential component of the let-7 microRNA pathway, and in conjunction with other factors, it may play a role in the loading of let-7 microRNA into the silencing complex.

Linkage between Substrate Recognition and Catalysis during Cleavage of Sarcin/Ricin Loop RNA by Restrictocin

Restrictocin is a site-specific endoribonuclease that inactivates ribosomes by cleaving the sarcin/ricin loop (SRL) of 23S-28S rRNA. Here we present a kinetic and thermodynamic analysis of the SRL cleavage reaction based on monitoring the cleavage of RNA oligonucleotides (2-27-mers). Restrictocin binds to a 27-mer SRL model substrate (designated wild-type SRL) via electrostatic interactions to form a nonspecific ground state complex E:S. At pH 6.7, physical steps govern the reaction rate: the wild-type substrate reacts at a partially diffusion-limited rate, and a faster-reacting SRL, containing a 3'-sulfur atom at the scissile phosphate, reacts at a fully diffusion-limited rate (k2/K1/2 = 1.1 x 10(9) M-1 s-1). At pH 7.4, the chemical step apparently limits the SRL cleavage rate. After the nonspecific binding step, restrictocin recognizes the SRL structure, which imparts 4.3 kcal/mol transition state stabilization relative to a single-stranded RNA. The two conserved SRL modules, bulged-G motif and GAGA tetraloop, contribute at least 2.4 and 1.9 kcal/mol, respectively, to the recognition. These findings suggest a model of SRL recognition in which restrictocin contacts the GAGA tetraloop and the bulged guanosine of the bulged-G motif to progress from the nonspecific ground state complex (E:S) to the higher-energy-specific complex (E.S) en route to the chemical transition state. Comparison of restrictocin with other ribonucleases revealed that restrictocin exhibits a 10(3)-10(6)-fold smaller ribonuclease activity against single-stranded RNA than do the restrictocin homologues, non-structure-specific ribonucleases T1 and U2. Together, these findings show how structural features of the SRL substrate facilitate catalysis and provide a mechanism for distinguishing between cognate and noncognate RNA.

A dynamic bulge in the U6 RNA internal stem–loop functions in spliceosome assembly and activation

The highly conserved internal stem-loop (ISL) of U6 spliceosomal RNA is unwound for U4/U6 complex formation during spliceosome assembly and reformed upon U4 release during spliceosome activation. The U6 ISL is structurally similar to Domain 5 of group II self-splicing introns, and contains a dynamic bulge that coordinates a Mg++ ion essential for the first catalytic step of splicing. We have analyzed the causes of growth defects resulting from mutations in the Saccharomyces cerevisiae U6 ISL-bulged nucleotide U80 and the adjacent C67-A79 base pair. Intragenic suppressors and enhancers of the cold-sensitive A79G mutation, which replaces the C-A pair with a C-G pair, suggest that it stabilizes the ISL, inhibits U4/U6 assembly, and may also disrupt spliceosome activation. The lethality of mutations C67A and C67G results from disruption of base-pairing potential between U4 and U6, as these mutations are fully suppressed by compensatory mutations in U4 RNA. Strikingly, suppressor analysis shows that the lethality of the U80G mutation is due not only to formation of a stable base pair with C67, as previously proposed, but also another defect. A U6-U80G strain in which mispairing with position 67 is prevented grows poorly and assembles aberrant spliceosomes that retain U1 snRNP and fail to fully unwind the U4/U6 complex at elevated temperatures. Our data suggest that the U6 ISL bulge is important for coupling U1 snRNP release with U4/U6 unwinding during spliceosome activation.

Identification of Tau Stem Loop RNA Stabilizers

Alternative splicing of tau exon 10 produces tau isoforms with either 3 (3R) or 4 (4R) repeated microtubule-binding domains. Increased ratios of 4R to 3R tau expression, above the physiological 1:1, leads to neurofibrillary tangles and causes neurodegenerative disease. An RNA stem loop structure plays a significant role in determining the ratio, with decreasing stability correlating with an increase in 4R tau mRNA expression. Recent studies have shown that aminoglycosides are able to bind and stabilize the tau stem loop in vitro, suggesting that other druglike small molecules could be identified and that such molecules might lead to decreased exon 10 splicing in vivo. The authors have developed a fluorescent high-throughput fluorescent binding assay and screened a library of approximately 110,000 compounds to identify candidate drugs that will bind the tau stem loop in vitro. In addition, they have developed a fluorescent-based RNA probe to assay the stabilizing effects of candidate drugs on the tau stem loop RNA. These assays should be applicable to the general problem of identifying small molecules that interact with mRNA secondary structures.

Solid-phase synthesis and thermal denaturation study of cyclic PNAs targeting the HIV-1 TAR RNA loop

Cyclic PNAs targeting the HIV-1 TAR RNA loop have been synthesized following a convenient solid-phase strategy which allows on-resin cyclisation. UV-monitored thermal denaturation studies demonstrate that these cyclic PNAs are able to strongly interact with their TAR RNA target, very likely through the formation of a six-base pair stable complex, involving the TAR RNA loop.