Presence Of RNase Research Articles

Genetically Encoded, Functional Single-Strand RNA Origami: Anticoagulant.

Nucleic acid aptamers selected for thrombin binding have been previously shown to possess anticoagulant activity; however, problems with rapid renal clearance and short circulation half-life have prevented translation to clinical usefulness. Here, a family of self-folding, functional RNA origami molecules bearing multiple thrombin-binding RNA aptamers and showing significantly improved anticoagulant activity is described. These constructs may overcome earlier problems preventing clinical use of nucleic acid anticoagulants. RNA origami structures are designed in silico and produced by in vitro transcription from DNA templates. Incorporation of 2'-fluoro-modified C- and U-nucleotides is shown to increase nuclease resistance and stability during long-term storage. Specific binding to human thrombin as well as high stability in the presence of RNase A and in human plasma, comparatively more stable than DNA is demonstrated. The RNA origami constructs show anticoagulant activity sevenfold greater than free aptamer and higher than previous DNA weave tiles decorated with DNA aptamers. Anticoagulation activity is maintained after at least 3 months of storage in buffer at 4 °C. Additionally, inhibition of thrombin is shown to be reversed by addition of single-stranded DNA antidotes. This project paves the way for development of RNA origami for potential therapeutic applications especially as a safer surgical anticoagulant.

Apoptosis of A549 cells by small interfering RNA targeting survivin delivery using poly-β-amino ester/guanidinylated O-carboxymethyl chitosan nanoparticles

Gene-based therapeutics has emerged as a promising approach for human cancer therapy. Among a variety of non-viral vectors, polymer vectors are particularly attractive due to their safety and multivalent groups on their surface. This study focuses on guanidinylated O-carboxymethyl chitosan (GOCMCS) along with poly-β-amino ester(PBAE) for siRNA delivery. Binding efficiency of PBAE/siRNA/GOCMCS nanoparticles were characterized by gel electrophoresis. The siRNA-loaded nanoparticles were found to be stable in the presence of RNase A, serum and BALF respectively. Fine particle fraction (FPF) which was determined by a two-stage impinger (TSI) was 57.8% ± 2.6%. The particle size and zeta potential of the nanoparticles were 153.8 ± 12.54 nm and + 12.2 ± 4.94 mV. In vitro cell transfection studies were carried out with A549 cells. The cellular uptake was significantly increased. When the cells were incubated with siSurvivin-loaded nanoparticles, it could induce 26.83% ± 0.59% apoptosis of A549 cells and the gene silencing level of survivin expression in A549 cells were 30.93% ± 2.27%. The results suggested that PBAE/GOCMCS nanoparticle was a very promising gene delivery carrier.

New platinum(II) and palladium(II) complexes with substituted terpyridine ligands: synthesis and characterization, cytotoxicity and reactivity towards biomolecules.

A series of palladium(II) (1-3) and platinum(II) chloride complexes (4 and 5) with 2,2':6',2″-terpyridine (terpy) derivatives substituted at the 4' position, was synthesized and fully characterized. Single crystal X-ray diffraction analysis of complexes 2, 3 and 5 showed tridentate coordination of the 4'-substituted terpyridine (terpy) ligands to the metal center. Moreover, in vitro cytotoxic activity of these complexes toward a panel of human cancer cell lines (lung cancer A549, colorectal cancer HCT116, ovarian cancer IGROV-1) and toward normal cell line HDF (dermal fibroblast) was determined by Trypan Blue exclusion assay. Overall, the tested compounds manifested a relevant cytotoxicity for the selected cancer cell lines with complex 4 also showing a modest cytotoxicity on the normal cell lines. To better understand the mode of action of these metal complexes, their reactivity with three model proteins, i.e. hen egg white lysozyme (HEWL), cytochrome c (cyt c) and ribonuclease A (RNase A) were comparatively investigated through ESI-MS analysis. The results highlighted a different behavior between the two series of complexes being platinum compounds more reactive toward RNase and cyt c than palladium compounds. Based on the obtained results, it is proposed that in presence of RNase A and cyt c, the platinum complexes undergo activation through release of labile ligands followed by binding to the protein. In contrast, palladium complexes revealed a far lower reactivity implying the likely occurrence of a different mechanism of action.

Peptide-oligonucleotide conjugates exhibiting pyrimidine-X cleavage specificity efficiently silence miRNA target acting synergistically with RNase H

Taking into account the important role of miRNA in carcinogenesis, oncogenic miRNAs are attractive molecules for gene-targeted therapy. Here, we developed a novel series of peptide-oligonucleotide conjugates exhibiting ribonuclease activity targeted to highly oncogenic miRNAs miR-21 and miR-17. When designing the conjugates, we enhanced both nuclease resistance of the targeted oligodeoxyribonucleotide by introducing at its 3′-end mini-hairpin structure displaying high thermostability and robustness against nuclease digestion and the efficiency of its functioning by attachment of the catalytic construction (amide)NH2-Gly(ArgLeu)4-TCAA displaying ribonuclease activity to its 5′-end. Designed miRNases efficiently cleaved miRNA targets, exhibiting Pyr-X specificity, and cleavage specificity had strong dependence on the miRNA sequence in the site of peptide location. In vitro, designed miRNases do not prevent cleavage of miRNA bound with the conjugate by RNase H, and more than an 11-fold enhancement of miRNA cleavage by the conjugate is observed in the presence of RNase H. In murine melanoma cells, miRNase silences mmu-miR-17 with very high efficiency as a result of miR-17 cleavage by miRNase and by recruited RNase H. Thus, miRNases provide a system of double attack of the miRNA molecules, significantly increasing the efficiency of miRNA downregulation in the cells in comparison with antisense oligonucleotide.

Dendrimer as a new potential carrier for topical delivery of siRNA: A comparative study of dendriplex vs. lipoplex for delivery of TNF-α siRNA

Topical delivery of siRNA is challenging task due to complex barrier property of stratum corneum and cationic lipid based carriers have been widely explored for this purpose due to improved permeation through skin. For gene delivery application, dendrimers are considered as efficient carrier due to their cationic nature and well-defined surface groups. However, they are not well explored for topical delivery. This work compares the suitability of PAMAM dendrimer with DOTAP liposome for topical delivery of siRNA against TNF-α. The particle size, zeta potential and entrapment efficiency of dendriplex were 99.80 ± 1.80 nm, 13.40 ± 4.84 mV and 98.72 ± 2.02% whereas for lipoplex were 174.80 ± 0.80 nm, 29.96 ± 0.51 mV and 94.99 ± 5.01% respectively. Both the formulations were stable in serum and in the presence of RNAse. TNF-α is inflammatory cytokine, hence the in vivo efficacy of developed formulations was determined using psoriatic plaque model. Results suggested improved phenotypic and histopathological features and reduced levels of IL-6, TNF-α, IL-17 and IL-22 for dendriplex and lipoplex treated groups in comparison to Imiquimod treated group. These findings suggest that dendrimer can be a potential carrier for topical gene delivery.

Double determination of long noncoding RNAs from lung cancer via multi-amplified electrochemical genosensor at sub-femtomole level

An ultrasensitive electrochemical genosensor has been fabricated for the double determination of two different specific sequences deduced from the maternally expressed gene3 (MEG3) lncRNA (long noncoding RNA), which was demonstrated by coupling RNase A-aided target recycling with DNA supersandwich-induced signal enhancement, based on a composite interface of graphene-like tungsten disulfide/dendritic gold nanostructures (WS2/DGN). Firstly, duple target sequences of T1 and T2 were captured by the primer probes of P1/P2 functionalized Fe3O4@C magnetic nanoparticles, via the DNA/RNA hybridization between T1/T2 and P1/P2. In the presence of RNase A, T1 and T2 were released to trigger the target recycling, accompanied by the generation of numerous intermediate DNAs designated as IT1 and IT2, respectively. After the magnetic separation, the IT1 and IT2 were liberated and hybridized with the capture probes of CP1/CP2 loaded DGN/WS2 modified electrode. Subsequently, the stepwise DNA hybridization chain reactions (HCR) labeled with ferrocene (Fc) and methyleneblue (MB) were processed, respectively. The DPV current values of Fc and MB were recorded, which were proportional with the concentration of T1 and T2, respectively. Using the multiplexed amplification strategy, this newly designed genosensor provided a wide linear range from 1 fM to 100 pM with a low detection limit of 0.25 fM for T1 and 0.3 fM for T2. The application of the genosensor in real serum sample has also been studied, confirming the excellent selectivity and sensitivity for the application in bioanalysis and clinical diagnostics.

A Role For The Transcription Factor SNAIL In Alternative Splicing

The transcription factor SNAIL is one of the key regulators of an early developmental process called the Epithelial to Mesenchymal Transition. This process is characterized by a cell‐state change from a highly differentiated, epithelial phenotype to a less differentiated, migratory mesenchymal phenotype. SNAIL binds to E‐box motifs in promoter regions and recruits chromatin remodelers to repress or activate gene expression. To identify proteins associated with SNAIL, we used co‐immunoprecipitation of SNAIL‐bound proteins followed by mass spectrometry. A large number of splicing proteins associated with SNAIL, including SRSF1, a regulator of alternative splicing.Co‐immunofluorescence and co‐immunoprecipitation data from our lab indicate that SNAIL associates with splicing protein SRSF1. Further, SNAIL co‐immunoprecipitated with SRSF1 in the presence of DNase, but not in the presence of RNase, indicating that the SNAIL‐SRSF1 association is RNA‐dependent. Knockdown of SNAIL with siRNA resulted in loss of mesenchymal splice isoforms and reversion to epithelial specific isoforms. RNA Immunoprecipitation of SNAIL‐ and SRSF1‐ associated RNAs showed increased enrichment for mesenchymal specific isoforms of mRNAs, suggesting that SNAIL and SRSF1 influence alternative splicing.We hypothesize that SNAIL regulates alternative splicing by binding to RNA and recruiting SRSF1. We are currently investigating genome‐wide changes in alternative splicing regulated by SNAIL, and working to understand the mechanism by which SNAIL interaction with the spliceosome leads to alternative splicing. As transcription and splicing are closely coupled, we predict that transcription factor regulation of splicing through RNA binding is universally utilized by other biological systems to alter phenotype in response to external stimuli.Support or Funding InformationUniversity of North Dakota, NIH Grant #1P30GM103329 and NIH COBRE #P20GM104360‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Design and Development of a Quantitative TaqMan Real-Time PCR Assay for Evaluation of HIV-1 (group M) Viral Load in Plasma Using Armored RNA Standard.

Human immunodeficiency virus-1 (HIV-1) is a viral infectious agent that gradually extinguishes the immune system, resulting in the acquired immune deficiency syndrome (AIDS). The aim of this study was to develop a TaqMan based detection assay to evaluate HIV-1 plasma viral load and to construct a stable internal positive control (IPC) and external positive control (EPC) RNA based on Armored RNA (AR) technology. The MS2 maturase, coat protein gene and HIV-1 pol gene were cloned in pET-32a plasmid. The recently fabricated recombinant plasmid was transformed into Escherichia coli strain BL2 (DE3) and protein expression and Armored RNA was fabricated in presence of isopropyl-L-thio-D-galactopyranoside (IPTG). The Armored RNA was precipitated and purified by polyethylene glycol (PEG) and sephacryl S-200 chromatography. The stability of Armored RNA was evaluated by treatment with DNase I and RNase A and confirmed by transmission electron microscopy (TEM) and gel agarose electrophoresis. The specificity, sensitivity, inter- and intra-day precision, and the dynamic range of the assay were experimentally determined. The AR was stable in presence of ribonuclease, and the assay had a dynamic detection range from 101 to 105 copies of AR. The coefficient of variation (CV) was 4.8% for intra-assay and 5.8% for inter-assay precision. Clinical specificity and sensitivity of the assay were assessed at 100% and 96.66%, respectively. The linear regression analysis confirmed a high correlation between the in-house and the commercial assay, Real Star HIV-1-qRTPCR, respectively (R2 = 0.888). The AR standard is non-infectious and highly resistant in the presence of ribonuclease. The TaqMan assay developed is able to quantify HIV viral load based on a novel conserved region of HIV-1 pol gene which has minimal sequence inconsistency.

Degradation of RNA during lysis of Escherichia coli cells in agarose plugs breaks the chromosome.

The nucleoid of Escherichia coli comprises DNA, nucleoid associated proteins (NAPs) and RNA, whose role is unclear. We found that lysing bacterial cells embedded in agarose plugs in the presence of RNases caused massive fragmentation of the chromosomal DNA. This RNase-induced chromosomal fragmentation (RiCF) was completely dependent on the presence of RNase around lysing cells, while the maximal chromosomal breakage required fast cell lysis. Cell lysis in plugs without RNAse made the chromosomal DNA resistant to subsequent RNAse treatment. RiCF was not influenced by changes in the DNA supercoiling, but was influenced by growth temperature or age of the culture. RiCF was partially dependent on H-NS, histone-like nucleoid structuring- and global transcription regulator protein. The hupAB deletion of heat-unstable nucleoid protein (HU) caused increase in spontaneous fragmentation that was further increased when combined with deletions in two non-coding RNAs, nc1 and nc5. RiCF was completely dependent upon endonuclease I, a periplasmic deoxyribonuclease that is normally found inhibited by cellular RNA. Unlike RiCF, the spontaneous fragmentation in hupAB nc1 nc5 quadruple mutant was resistant to deletion of endonuclease I. RiCF-like phenomenon was observed without addition of RNase to agarose plugs if EDTA was significantly reduced during cell lysis. Addition of RNase under this condition was synergistic, breaking chromosomes into pieces too small to be retained by the pulsed field gels. RNase-independent fragmentation was qualitatively and quantitatively comparable to RiCF and was partially mediated by endonuclease I.

Determination of RNase H activity via real-time monitoring of target-triggered rolling circle amplification.

The authors describe a method for real-time monitoring of the activity of ribonuclease H (RNase H). It is based on target-triggered rolling circle amplification (RCA). It utilizes a specially designed primer that contains a RNA sequence in the center and an amino group at the 3'-end. In the absence of RNase H, the primerwhen hybridized to a circular DNA template is not extended by DNA polymerase due to the amino group at the 3'-end. In contrast, the presence of RNase H specifically degrades the RNA sequence of the primer hybridized to the circular DNA template. This results in the conversion of the 3'-amino group to a 3'-hydroxy group and thereby enables the extension reaction promoted by DNA polymerase. This, consequently, leads to efficient RCA producing a long concatenated DNA strand. Its generation can be monitored in real-time by using the fluorescent dye SYBR green II which is specific for single-stranded DNA. Based on this RNase H-triggered RCA, RNase H activity can be selectively determined at levels as low as 0.019U·mL-1 with a total assay time of <5min. The diagnostic capability of this assay was demonstrated by monitoring the activity of RNase H in tumor cells. Graphical abstract Schematic of real-time monitoring of ribonuclease H (RNase H) activity based on target-triggered rolling circle amplification (RCA). RNase H that degrades RNA in primer, converts 3'-amino group to 3'-hydroxy group, which promotes RCA with fluorescence enhancement of the probe SYBR green II.

Influence of mismatched and bulged nucleotides on SNP-preferential RNase H cleavage of RNA-antisense gapmer heteroduplexes

This study focused on determining design rules for gapmer-type antisense oligonucleotides (ASOs), that can differentiate cleavability of two SNP variants of RNA in the presence of ribonuclease H based on the mismatch type and position in the heteroduplex. We describe the influence of structural motifs formed by several arrangements of multiple mismatches (various types of mismatches and their position within the ASO/target RNA duplex) on RNase H cleavage selectivity of five different SNP types. The targets were mRNA fragments of APP, SCA3, SNCA and SOD1 genes, carrying C-to-G, G-to-C, G-to-A, A-to-G and C-to-U substitutions. The results show that certain arrangements of mismatches enhance discrimination between wild type and mutant SNP alleles of RNA in vitro as well as in HeLa cells. Among the over 120 gapmers tested, we found two gapmers that caused preferential degradation of the mutant allele APP 692 G and one that led to preferential cleavage of the mutant SNCA 53 A allele, both in vitro and in cells. However, several gapmers promoted selective cleavage of mRNA mutant alleles in in vitro experiments only.

Phorbol-12-myristate-13-acetate-mediated stabilization of leukemia inhibitory factor (lif) mRNA: involvement of Nucleolin and PCBP1.

Leukemia inhibitory factor (LIF) is a potent pleiotropic cytokine involved in diverse biological activities, thereby requiring precise spatial and temporal control of its expression. The present study reveals that enhanced expression of LIF in response to PMA (phorbol-12-myristate-13-acetate) in human histiocytic lymphoma cell line U937 largely happens through stabilization of its mRNA. Functional characterization of the long 3'-untranslated region of human lif mRNA revealed several conserved sequences with conventional cis-acting elements. A 216 nucleotide containing proximal cis-element with two AUUUA pentamers and four poly-rC sequences demonstrated significant mRNA destabilizing potential, which, on treatment with PMA, showed stabilizing activity. Affinity chromatography followed by western blot and RNA co-immunoprecipitation of PMA-treated U937 extract identified Nucleolin and PCBP1 as two protein trans-factors interacting with lif mRNA, specifically to the proximal non-conventional AU-rich region. PMA induced nucleo-cytoplasmic translocation of both Nucleolin and PCBP1. RNA-dependent in vivo co-association of both these proteins with lif mRNA was demonstrated by decreased co-precipitation in the presence of RNase. Ectopic overexpression of Nucleolin showed stabilization of both intrinsic lif mRNA and gfp reporter, whereas knockdown of Nucleolin and PCBP1 demonstrated a significant decrease in both lif mRNA and protein levels. Collectively, this report establishes the stabilization of lif mRNA by PMA, mediated by the interactions of two RNA-binding proteins, Nucleolin and PCBP1 with a proximal cis-element.

Human DMBT1-Derived Cell-Penetrating Peptides for Intracellular siRNA Delivery.

Small interfering RNA (siRNA) is a promising molecule for gene therapy, but its therapeutic administration remains problematic. Among the recently proposed vectors, cell-penetrating peptides show great promise in in vivo trials for siRNA delivery. Human protein DMBT1 (deleted in malignant brain tumor 1) is a pattern recognition molecule that interacts with polyanions and recognizes and aggregates bacteria. Taking advantage of these properties, we investigated whether specific synthetic DMBT1-derived peptides could be used to formulate nanoparticles for siRNA administration. Using an electrophoretic mobility shift assay and UV spectra, we identified two DMBT1 peptides that could encapsulate the siRNA with a self- and co-assembly mechanism. The complexes were stable for at least 2 hr in the presence of either fetal bovine serum (FBS) or RNase A, with peptide-dependent time span protection. ζ-potential, circular dichroism, dynamic light scattering, and transmission electron microscopy revealed negatively charged nanoparticles with an average diameter of 10–800 nm, depending on the reaction conditions, and a spherical or rice-shaped morphology, depending on the peptide and β-helix conformation. We successfully transfected human MCF7 cells with fluorescein isothiocyanate (FITC)-DMBT1-peptide-Cy3-siRNA complexes. Finally, DMBT1 peptides encapsulating an siRNA targeting a fluorescent reporter gene showed efficient gene silencing in MCF7-recombinant cells. These results lay the foundation for a new research line to exploit DMBT1-peptide nanocomplexes for therapeutic siRNA delivery.

Polyvinylsulfonic acid: A Low-cost RNase inhibitor for enhanced RNA preservation and cell-free protein translation

ABSTRACTThe effectiveness and economics of polyvinyl sulfonic acid (PVSA) as a ribonuclease inhibitor for in vitro systems is reported. PVSA was shown to inhibit RNA cleavage in the presence of RNase A as well as in the presence of Escherichia coli lysate, suggesting that PVSA can act as a broader ribonuclease inhibitor. In addition, PVSA was shown to improve the integrity of mRNA transcripts by up to 5-fold in vitro as measured by their translational viability. Improved preservation of mRNA transcripts in the presence of PVSA under common RNA storage conditions is also reported. A cost comparison with commercially available RNAse inhibitors indicates the economic practicality of PVSA which is approximately 1,700 times less expensive than commonly used ribonuclease inhibitors. PVSA can also be separated from RNA by alcohol precipitation for applications that may be sensitive to the presence of PVSA.

Strong transcription blockage mediated by R-loop formation within a G-rich homopurine-homopyrimidine sequence localized in the vicinity of the promoter.

Guanine-rich (G-rich) homopurine–homopyrimidine nucleotide sequences can block transcription with an efficiency that depends upon their orientation, composition and length, as well as the presence of negative supercoiling or breaks in the non-template DNA strand. We report that a G-rich sequence in the non-template strand reduces the yield of T7 RNA polymerase transcription by more than an order of magnitude when positioned close (9 bp) to the promoter, in comparison to that for a distal (∼250 bp) location of the same sequence. This transcription blockage is much less pronounced for a C-rich sequence, and is not significant for an A-rich sequence. Remarkably, the blockage is not pronounced if transcription is performed in the presence of RNase H, which specifically digests the RNA strands within RNA–DNA hybrids. The blockage also becomes less pronounced upon reduced RNA polymerase concentration. Based upon these observations and those from control experiments, we conclude that the blockage is primarily due to the formation of stable RNA–DNA hybrids (R-loops), which inhibit successive rounds of transcription. Our results could be relevant to transcription dynamics in vivo (e.g. transcription ‘bursting’) and may also have practical implications for the design of expression vectors.

Shockwaves prevent from heart failure after acute myocardial ischaemia via RNA/protein complexes.

Shock wave treatment (SWT) was shown to induce regeneration of ischaemic myocardium via Toll‐like receptor 3 (TLR3). The antimicrobial peptide LL37 gets released by mechanical stress and is known to form complexes with nucleic acids thus activating Toll‐like receptors. We suggested that SWT in the acute setting prevents from the development of heart failure via RNA/protein release. Myocardial infarction in mice was induced followed by subsequent SWT. Heart function was assessed 4 weeks later via transthoracic echocardiography and pressure–volume measurements. Human umbilical vein endothelial cells (HUVECs) were treated with SWT in the presence of RNase and proteinase and analysed for proliferation, tube formation and LL37 expression. RNA release and uptake after SWT was evaluated. We found significantly improved cardiac function after SWT. SWT resulted in significantly higher numbers of capillaries and arterioles and less left ventricular fibrosis. Supernatants of treated cells activated TLR3 reporter cells. Analysis of the supernatant revealed increased RNA levels. The effect could not be abolished by pre‐treatment of the supernatant with RNase, but only by a sequential digestion with proteinase and RNase hinting strongly towards the involvement of RNA/protein complexes. Indeed, LL37 expression as well as cellular RNA uptake were significantly increased after SWT. We show for the first time that SWT prevents from left ventricular remodelling and cardiac dysfunction via RNA/protein complex release and subsequent induction of angiogenesis. It might therefore develop a potent regenerative treatment alternative for ischaemic heart disease.

Expression profile of circulating serum microRNAs in dogs with lymphoma

Serum microRNAs (miRNAs) are mediators of cell-to-cell communication and alter the cellular microenvironment; they are stable for hours under certain conditions in body fluids despite the presence of RNases. Certain miRNAs have been found to be altered in the serum or plasma of humans with various cancers and may represent promising, non-invasive biomarkers for various diseases in humans and animals. The objective of this study was to determine the expression profile of circulating miRNAs in the serum of dogs with lymphoma. Serum samples were obtained from 61 dogs with lymphoma and 40 control dogs, and real-time reverse transcription–polymerase chain reaction was used for miRNA measurement. In order to select candidate genes, a comprehensive expression analysis was undertaken prior to validation of several candidate miRNAs.Of 277 miRNAs, five (let-7b, miR-223, miR-25, miR-92a, and miR-423a) were selected as candidates. The expression levels of four miRNAs (let-7b, miR-223, miR-25, miR-92a) were significantly reduced in the lymphoma group, whereas miR-423a levels were significantly increased compared to the controls. When the lymphoma cases were categorized into high- or low-grade as well as into their anatomic form, miR-25 levels were lower in the serum samples from the lymphoma group compared to those from the control group. Although the biological function of serum miRNAs still remains unclear, determining their functional roles in serum and tissues will contribute not only to the identification of potential biomarkers but also to the elucidation of the pathogenesis of canine lymphoma.

A Single Molecule Perspective of Elongation by RNA Polymerase I

RNA polymerase I (Pol I) transcribes ribosomal DNA and is responsible for more than 60% of total transcription in a growing cell. Despite its central role in transcription and fundamental role in cell growth and proliferation, a detailed understanding of kinetics of transcription by Pol I is lacking. We present the first direct characterization of Pol I transcription elongation at the single molecule level. Tethered particle microscopy shows that the pause-free rate measured for Pol I, approximately 50 nt/s, is comparable to the in vivo rate estimated from the number of active genes, the cell division rate and the number of engaged polymerases determined from EM images. Furthermore, comparison of elongation traces in the presence or absence of RNAse A & T1, reveals strong interactions between the nascent RNA and the DNA tether that might modulate transcription elongation in vivo. In the presence of RNAse, Pol I processivity is higher than in the absence of RNAse. Furthermore, when the nascent transcript is not digested by RNAse, large tether length fluctuations are noticed which yield an apparently slower average elongation rate of approximately 20 nt/s.

Ligand-dependent exponential amplification of self-replicating RNA enzymes.

A general analytical method for the detection of target ligands has been developed, based on a special class of self-replicating aptazymes. These "autocatalytic aptazymes" are generated by linking an aptamer domain to the catalytic domain of a self-replicating RNA enzyme. Ligand-dependent self-replication of RNA proceeds in a self-sustained manner, undergoing exponential amplification at a constant temperature without the assistance of any proteins or other biological materials. The rate of exponential amplification is dependent on the concentration of the ligand, thus enabling quantitative ligand detection. This system has the potential to detect any ligand that can be recognized by an aptamer, including small molecules and proteins. The instability of RNA in biological samples due to the presence of ribonucleases can be overcome by employing the enantiomeric L-RNA form of the self-replicating enzyme. Methods for real-time fluorescence monitoring over the course of exponential amplification are currently being developed.

Biological stability and activity of siRNA in ionic liquids.

We report substantially enhanced biological stability of siRNA in a hydrated ionic liquid (IL) based on buffered choline dihydrogen phosphate (CDP). The results show that hydrated CDP IL substantially prolongs the shelf-life of siRNA for up to three months in the presence of RNase A. Remarkably, siRNA stored in the IL remained active and eGFP siRNA exhibited clear gene knockdown effects in eGFP-expressing HeLa cells.