Biomedical subjects

Mark A Behlke

Publications and source records attributed to Mark A Behlke.

17 recordsLinked to original sources

PubMed2006

Design of LNA probes that improve mismatch discrimination.

Locked nucleic acids (LNA) show remarkable affinity and specificity against native DNA targets. Effects of LNA modifications on mismatch discrimination were studied as a function of sequence context and identity of the mismatch using ultraviolet (UV) melting experiments. A triplet of LNA residues centered on the mismatch was generally found to have the largest discriminatory power. An exception was observed for G-T mismatches, where discrimination decreased when the guanine nucleotide at the mismatch site or even the flanking nucleotides were modified. Fluorescence experiments using 2-aminopurine suggest that LNA modifications enhance base stacking of perfectly matched base pairs and decrease stabilizing stacking interactions of mismatched base pairs. LNAs do not change the amount of counterions (Na+) that are released when duplexes denature. New guidelines are suggested for design of LNA probes, which significantly improve mismatch discrimination in comparison with unmodified DNA probes.

2-Aminopurine↗

PubMed2006

A structural basis for discriminating between self and nonself double-stranded RNAs in mammalian cells.

Nonspecific effects triggered by small interfering RNAs (siRNAs) complicate the use of RNA interference (RNAi) to specifically downregulate gene expression. To uncover the basis of these nonspecific activities, we analyzed the effect of chemically synthesized siRNAs on mammalian double-stranded RNA (dsRNA)-activated signaling pathways. siRNAs ranging from 21 to 27 nucleotides (nt) in length activated the interferon system when they lacked 2-nt 3' overhangs, a characteristic of Dicer products. We show that the recognition of siRNAs is mediated by the RNA helicase RIG-I and that the presence of 3' overhangs impairs its ability to unwind the dsRNA substrate and activate downstream signaling to the transcription factor IRF-3. These results suggest a structural basis for discrimination between microRNAs that are endogenous Dicer products, and nonself dsRNAs such as by-products of viral replication. These findings will enable the rational design of siRNAs that avoid nonspecific effects or, alternatively, that induce bystander effects to potentially increase the efficacy of siRNA-based treatments of viral infections or cancer.

Animals↗

PubMed2006

Stable expression of shRNAs in human CD34+ progenitor cells can avoid induction of interferon responses to siRNAs in vitro.

RNA interference occurs when cytoplasmic small interfering RNAs (siRNAs) enter the RNA-induced silencing complex and one strand guides cleavage of the target RNA by the Argonaute 2 protein. A significant concern when applying siRNAs or expressing small hairpin RNAs (shRNAs) in human cells is activation of the interferon (IFN) response. Synthetic siRNAs harboring certain motifs can induce an immune response when delivered to mouse and human immune cells such as peripheral blood mononuclear cells, monocytes, plasmacytoid dendritic cells (pDCs) and nonplasmacytoid dendritic cells (mDCs). In the present study we have tested the immunostimulatory effects of lipid-delivered siRNAs versus Pol III promoter-expressed shRNAs in primary CD34+ progenitor-derived hematopoietic cells. We show that in this system, lipid-delivered siRNAs are potent inducers of IFNalpha and type I IFN gene expression, whereas the same sequences when expressed endogenously are nonimmunostimulatory.

Antigens, CD34↗

PubMed2006

Progress towards in vivo use of siRNAs.

RNA interference (RNAi) has become the method of choice to suppress gene expression in vitro. It is also emerging as a powerful tool for in vivo research with over 90 studies published using synthetic small interfering RNAs in mammals. These reports demonstrate the potential for use of synthetic small interfering RNAs (siRNAs) as therapeutic agents, especially in the areas of cancer and viral infection. The number of reports using siRNAs for functional genomics applications, for validation of targets for small-molecule drug development programs, and to address questions of basic biology will rapidly grow as methods and protocols for use in animals become more established. This review will first discuss aspects of RNAi biochemistry and biology that impact in vivo use, especially as relates to experimental design, and will then provide an overview of published work with a focus on methodology.

Animals↗

PubMed2006

Characterization of modified antisense oligonucleotides in Xenopus laevis embryos.

A wide variety of modified oligonucleotides have been tested as antisense agents. Each chemical modification produces a distinct profile of potency, toxicity, and specificity. Novel cationic phosphoramidate-modified antisense oligonucleotides have been developed recently that have unique and interesting properties. We compared the relative potency and specificity of a variety of established antisense oligonucleotides, including phosphorothioates (PS), 2'-O-methyl (2'OMe) RNAs, locked nucleic acids (LNAs), and neutral methoxyethyl (MEA) phosphoramidates with new cationic N,N-dimethylethylenediamine (DMED) phosphoramidate-modified antisense oligonucleotides. A series of oligonucleotides was synthesized that targeted two sites in the Xenopus laevis survivin gene and were introduced into Xenopus embryos by microinjection. Effects on survivin gene expression were examined using quantitative real-time PCR. Of the various modified oligonucleotide designs tested, LNA/PS chimeras (which showed the highest melting temperature) and DMED/phosphodiester chimeras (which showed protection of neighboring phosphate bonds) were potent in reducing gene expression. At 40 nM, overall specificity was superior for the LNA/PS-modified compounds compared with the DMED-modified oligonucleotides. However, at 400 nM, both of these compounds led to significant degradation of survivin mRNA, even when up to three mismatches were present in the heteroduplex.

Amides↗

PubMed2005

Respiratory syncytial virus nonstructural protein 2 specifically inhibits type I interferon signal transduction.

Human respiratory syncytial virus (RSV) inhibits type I interferon-induced gene expression by decreasing expression of signal transducer and activator of transcription (Stat)2. To identify the RSV protein that mediates effects on Stat2, airway epithelial cells were infected with vaccinia virus vectors that express single RSV proteins. Expression of RSV nonstructural (NS)2 protein alone was sufficient to decrease Stat2 levels. Furthermore, decreasing RSV NS2 levels using RNA interference in respiratory epithelial cells inhibited the RSV-mediated decrease in Stat2 expression. Airway epithelial cells were also infected with equivalent inoculums of RSV without or with single gene deletions of NS1 or NS2. RSV infection without NS2 expression did not result in decreased Stat2 levels or loss of type I interferon-dependent signaling, indicating that NS2 expression is necessary for RSV effects on Stat2. Taken together, our results indicate that NS2 regulates Stat2 levels during RSV infection, thereby modulating viral effects on interferon-dependent gene expression.

Cells, Cultured↗

PubMed2005

Functional polarity is introduced by Dicer processing of short substrate RNAs.

Synthetic RNA duplexes that are substrates for Dicer are potent triggers of RNA interference (RNAi). Blunt 27mer duplexes can be up to 100-fold more potent than traditional 21mer duplexes. Not all 27mer duplexes show increased potency. Evaluation of the products of in vitro dicing reactions using electrospray ionization mass spectrometry reveals that a variety of products can be produced by Dicer cleavage. Use of asymmetric duplexes having a single 2-base 3'-overhang restricts the heterogeneity that results from dicing. Inclusion of DNA residues at the ends of blunt duplexes also limits heterogeneity. Combination of asymmetric 2-base 3'-overhang with 3'-DNA residues on the blunt end result in a duplex form which directs dicing to predictably yield a single primary cleavage product. It is therefore possible to design a 27mer duplex which is processed by Dicer to yield a specific, desired 21mer species. Using this strategy, two different 27mers can be designed that result in the same 21mer after dicing, one where the 3'-overhang resides on the antisense (AS) strand and dicing proceeds to the 'right' ('R') and one where the 3'-overhang resides on the sense (S) strand and dicing proceeds to the 'left' ('L'). Interestingly, the 'R' version of the asymmetric 27mer is generally more potent in reducing target gene levels than the 'L' version 27mer. Strand targeting experiments show asymmetric strand utilization between the two different 27mer forms, with the 'R' form favoring S strand and the 'L' form favoring AS strand silencing. Thus, Dicer processing confers functional polarity within the RNAi pathway.

Cell Line↗

PubMed2005

Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability.

Single and dual-labeled fluorescent oligodeoxynucleotides are used in many molecular biology applications. We investigated the effects of commonly used fluorescent dyes and quenchers on the thermodynamic stability of a model probe-target DNA duplex. We demonstrate that those effects can be significant. Fluorescent dyes and quenchers were attached to the probe ends. In certain combinations, these groups stabilized the duplex up to 1.8kcal/mol and increased T(m) up to 4.3 degrees C. None of the groups tested significantly destabilized the duplex. Rank order of potency was, starting with the most stabilizing group: Iowa Black RQ approximately Black Hole 2>Cy5 approximately Cy3>Black Hole 1>QSY7 approximately Iowa Black FQ>Texas Red approximately TAMRA>FAM approximately HEX approximately Dabcyl>TET. Longer linkers decreased stabilizing effects. Hybridizations to targets with various dangling ends were also studied and were found to have only minor effects on thermodynamic stability. Depending on the dye/quencher combination employed, it can be important to include thermodynamic contributions from fluorophore and quencher when designing oligonucleotide probe assays.

Base Sequence↗

PubMed2005

A potential role for RNA interference in controlling the activity of the human LINE-1 retrotransposon.

Long interspersed nuclear elements (LINE-1 or L1) comprise 17% of the human genome, although only 80-100 L1s are considered retrotransposition-competent (RC-L1). Despite their small number, RC-L1s are still potential hazards to genome integrity through insertional mutagenesis, unequal recombination and chromosome rearrangements. In this study, we provide several lines of evidence that the LINE-1 retrotransposon is susceptible to RNA interference (RNAi). First, double-stranded RNA (dsRNA) generated in vitro from an L1 template is converted into functional short interfering RNA (siRNA) by DICER, the RNase III enzyme that initiates RNAi in human cells. Second, pooled siRNA from in vitro cleavage of L1 dsRNA, as well as synthetic L1 siRNA, targeting the 5'-UTR leads to sequence-specific mRNA degradation of an L1 fusion transcript. Finally, both synthetic and pooled siRNA suppressed retrotransposition from a highly active RC-L1 clone in cell culture assay. Our report is the first to demonstrate that a human transposable element is subjected to RNAi.

5' Untranslated Regions↗

PubMed2004

Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy.

RNA interference (RNAi) is the process of sequence-specific post-transcriptional gene silencing triggered by double-stranded RNAs. In attempts to identify RNAi triggers that effectively function at lower concentrations, we found that synthetic RNA duplexes 25-30 nucleotides in length can be up to 100-fold more potent than corresponding conventional 21-mer small interfering RNAs (siRNAs). Some sites that are refractory to silencing by 21-mer siRNAs can be effectively targeted by 27-mer duplexes, with silencing lasting up to 10 d. Notably, the 27-mers do not induce interferon or activate protein kinase R (PKR). The enhanced potency of the longer duplexes is attributed to the fact that they are substrates of the Dicer endonuclease, directly linking the production of siRNAs to incorporation in the RNA-induced silencing complex. These results provide an alternative strategy for eliciting RNAi-mediated target cleavage using low concentrations of synthetic RNA as substrates for cellular Dicer-mediated cleavage.

Gene Expression Regulation↗

PubMed2004

Effects of sodium ions on DNA duplex oligomers: improved predictions of melting temperatures.

Melting temperatures, T(m), were systematically studied for a set of 92 DNA duplex oligomers in a variety of sodium ion concentrations ranging from 69 mM to 1.02 M. The relationship between T(m) and ln [Na(+)] was nonlinear over this range of sodium ion concentrations, and the observed melting temperatures were poorly predicted by existing algorithms. A new empirical relationship was derived from UV melting data that employs a quadratic function, which better models the melting temperatures of DNA duplex oligomers as sodium ion concentration is varied. Statistical analysis shows that this improved salt correction is significantly more accurate than previously suggested algorithms and predicts salt-corrected melting temperatures with an average error of only 1.6 degrees C when tested against an independent validation set of T(m) measurements obtained from the literature. Differential scanning calorimetry studies demonstrate that this T(m) salt correction is insensitive to DNA concentration. The T(m) salt correction function was found to be sequence-dependent and varied with the fraction of G.C base pairs, in agreement with previous studies of genomic and polymeric DNAs. The salt correction function is independent of oligomer length, suggesting that end-fraying and other end effects have little influence on the amount of sodium counterions released during duplex melting. The results are discussed in the context of counterion condensation theory.

Base Pairing↗

PubMed2003

Thermodynamic treatment of oligonucleotide duplex-simplex equilibria.

Thermodynamic formulations have been devised to obtain Delta G degrees values directly from spectroscopic data at a fixed common temperature in nucleic acid duplex-simplex melting curves. In addition, the dependence of melting on salt concentration has been expressed in terms of a stepwise stoichiometric representation, which leads to a specific equation for the partition of the added sodium ions between the different oligonucleotide forms.

Betaine↗

PubMed2003

Spectroscopic features of dual fluorescence/luminescence resonance energy-transfer molecular beacons.

Molecular beacons have the potential to become a powerful tool in gene detection and quantification in living cells. Here we report a novel dual molecular beacons approach to reduce false-positive signals in detecting target nucleic acids in homogeneous assays. A pair of molecular beacons, each containing a fluorescence quencher and a reporter fluorophore, one with a donor and a second with an acceptor fluorophore, hybridize to adjacent regions on the same target resulting in fluorescence resonance energy transfer (FRET). The detection of a FRET signal leads to a substantially increased signal-to-background ratio compared with that seen in single molecular beacon assays and enables discrimination between fluorescence due to specific probe/target hybridization and a variety of possible false-positive events. Further, when a lanthanide chelate is used as a donor in a dual-probe assay, extremely high signal-to-background ratios can be achieved owing to the long lifetime and sharp emission peaks of the donor and the time-gated detection of acceptor fluorescence emission. These new approaches allow for the ultrasensitive detection of target molecules in a way that could be readily applied to real-time imaging of gene expression in living cells.

Base Sequence↗

PubMed2003

Hybridization of 2'-O-methyl and 2'-deoxy molecular beacons to RNA and DNA targets.

Molecular beacons are stem-loop hairpin oligonucleotide probes labeled with a fluorescent dye at one end and a fluorescence quencher at the other end; they can differentiate between bound and unbound probes in homogeneous hybridization assays with a high signal-to-background ratio and enhanced specificity compared with linear oligonucleotide probes. However, in performing cellular imaging and quantification of gene expression, degradation of unmodified molecular beacons by endogenous nucleases can significantly limit the detection sensitivity, and results in fluorescence signals unrelated to probe/target hybridization. To substantially reduce nuclease degradation of molecular beacons, it is possible to protect the probe by substituting 2'-O-methyl RNA for DNA. Here we report the analysis of the thermodynamic and kinetic properties of 2'-O-methyl and 2'-deoxy molecular beacons in the presence of RNA and DNA targets. We found that in terms of molecular beacon/target duplex stability, 2'-O-methyl/RNA > 2'-deoxy/RNA > 2'-deoxy/DNA > 2'-O-methyl/DNA. The improved stability of the 2'-O-methyl/RNA duplex was accompanied by a slightly reduced specificity compared with the duplex of 2'-deoxy molecular beacons and RNA targets. However, the 2'-O-methyl molecular beacons hybridized to RNA more quickly than 2'-deoxy molecular beacons. For the pairs tested, the 2'-deoxy-beacon/DNA-target duplex showed the fastest hybridization kinetics. These findings have significant implications for the design and application of molecular beacons.

DNA↗

PubMed2003

Hybridization kinetics and thermodynamics of molecular beacons.

Molecular beacons are increasingly being used in many applications involving nucleic acid detection and quantification. The stem-loop structure of molecular beacons provides a competing reaction for probe-target hybridization that serves to increase probe specificity, which is particularly useful when single-base discrimination is desired. To fully realize the potential of molecular beacons, it is necessary to optimize their structure. Here we report a systematic study of the thermodynamic and kinetic parameters that describe the molecular beacon structure-function relationship. Both probe and stem lengths are shown to have a significant impact on the binding specificity and hybridization kinetic rates of molecular beacons. Specifically, molecular beacons with longer stem lengths have an improved ability to discriminate between targets over a broader range of temperatures. However, this is accompanied by a decrease in the rate of molecular beacon-target hybridization. Molecular beacons with longer probe lengths tend to have lower dissociation constants, increased kinetic rate constants, and decreased specificity. Molecular beacons with very short stems have a lower signal-to-background ratio than molecular beacons with longer stems. These features have significant implications for the design of molecular beacons for various applications.

DNA Probes↗

PubMed2002

Hybridization of 2'-O-methyl and 2'-deoxy molecular beacons to RNA and DNA targets.

DNA↗

PubMed2002

Structure-function relationships of shared-stem and conventional molecular beacons.

Molecular beacons are oligonucleotide probes capable of forming a stem-loop hairpin structure with a reporter dye at one end and a quencher at the other end. Conventional molecular beacons are designed with a target-binding domain flanked by two complementary short arm sequences that are independent of the target sequence. Here we report the design of shared-stem molecular beacons with one arm participating in both stem formation when the beacon is closed and target hybridization when it is open. We performed a systematic study to compare the behavior of conventional and shared-stem molecular beacons by conducting thermodynamic and kinetic analyses. Shared-stem molecular beacons form more stable duplexes with target molecules than conventional molecular beacons; however, conventional molecular beacons may discriminate between targets with a higher specificity. For both conventional and shared-stem molecular beacons, increasing stem length enhanced the ability to differentiate between wild-type and mutant targets over a wider range of temperatures. Interestingly, probe-target hybridization kinetics were similar for both classes of molecular beacons and were influenced primarily by the length and sequence of the stem. These findings should enable better design of molecular beacons for various applications.

Kinetics↗