Efficient Incorporation of CoA, NAD and FAD Into RNA by \u3ci\u3eIn Vitro\u3c/i\u3e Transcription

Protein enzymes frequently recruit small molecule coenzymes to perform a variety of biochemical reactions. While the catalytic activities of RNA have been expanding rapidly, a similar strategy for RNA to utilize coenzymes and to increase its functional capabilities has yet to be demonstrated. A general in vitro transcription procedure has been developed to efficiently prepare RNA with coenzymes CoA, NAD and FAD covalently attached to the 5\u27 end. These adenosine-containing coenzymes initiate transcription under the T7 class II promoter by T7 RNA polymerase. In addition to the three coenzymes, other adenosine-containing molecules may be incorporated into the first nucleotide position of RNA as well. This method provides easy access to CoA-, NAD- and FAD-RNA, which may find broad applications in generating coenzyme- utilizing ribozymes. In addition, both oxidized FAD and reduced NADH are highly fluorescent. NADH-RNA and FAD-RNA can therefore be used as probes for DNA/RNA detection and for structural investigation of RNA function by fluorescence spectroscopy

Optimal Random Libraries For the Isolation of Catalytic RNA

The relationship between ribozyme size and catalytic activity is of fundamental importance for RNA catalysis and molecular evolution in the RNA world. We have performed a series of competitive in vitro selection experiments to probe the relationship using RNA libraries containing size-heterogeneous random regions. Our experiments have established an inverse correlation between RNA replication efficiency (the combined efficiency of PCR amplification, transcription, and reverse transcription) and RNA size. A number of ribozyme sequences have been isolated from different RNA size groups under competitive selection conditions. Comprehensive kinetic analysis on isolated ribozymes has revealed that large ribozymes do not confer a significant catalytic superiority over smaller ones under most selection conditions, and actually impose two significant problems of replication inefficiency and RNA misfolding into inactive conformations. The fraction of a misfolded ribozyme population is defined as alpha. Large ribozymes tend to possess high alpha values, which may significantly reduce ribozyme performance. Our results suggest that a random region of around 60 nucleotides represents the optimal balance between ribozyme catalytic activity, RNA misfolding (alpha), and replication efficiency, and may therefore constitute the most advantageous RNA libraries for successful isolation of functional RNA sequences. ©2005 Landes Bioscience

\u3ci\u3eIn Vivo\u3c/i\u3e Cloning of Up to 16 kb Plasmids in \u3ci\u3eE. Coli\u3c/i\u3e is As Simple As PCR

The precise assembly of defined DNA sequences into plasmids is an essential task in bioscience research. While a number of molecular cloning techniques have been developed, many methods require specialized expensive reagents or laborious experimental procedure. Not surprisingly, conventional cloning techniques based on restriction digestion and ligation are still commonly used in routine DNA cloning. Here, we describe a simple, fast, and economical cloning method based on RecA- and RecET-independent in vivo recombination of DNA fragments with overlapping ends using E. coli. All DNA fragments were prepared by a 2-consecutive PCR procedure with Q5 DNA polymerase and used directly for transformation resulting in 95% cloning accuracy and zero background from parental template plasmids. Quantitative relationships were established between cloning efficiency and three factors–the length of overlapping nucleotides, the number of DNA fragments, and the size of target plasmids–which can provide general guidance for selecting in vivo cloning parameters. The method may be used to accurately assemble up to 5 DNA fragments with 25 nt overlapping ends into relatively small plasmids, and 3 DNA fragments into plasmids up to 16 kb in size. The whole cloning procedure may be completed within 2 days by a researcher with little training in cloning. The combination of high accuracy and zero background eliminates the need for screening a large number of colonies. The method requires no enzymes other than Q5 DNA polymerase, has no sequence restriction, is highly reliable, and represents one of the simplest, fastest, and cheapest cloning techniques available. Our method is particularly suitable for common cloning tasks in the lab where the primary goal is to quickly generate a plasmid with a pre-defined sequence at low costs

Novel Cyanine-AMP Conjugates for Efficient 5\u27 RNA Fluorescent Labeling by One-Step Transcription and Replacement of [γ-\u3csup\u3e32\u3c/sup\u3eP] ATP in RNA Structural Investigation

Two novel fluorescent cyanine-AMP conjugates, F550/570 and F650/670, have been synthesized to serve as transcription initiators under the T7 φ2.5 promoter. Efficient fluorophore labeling of 5′ RNA is achieved in a single transcription step by including F550/570 and F650/670 in the transcription solution. The current work makes fluorescently labeled RNA readily available for broad applications in biochemistry, molecular biology, structural biology and biomedicine. In particular, site-specifically fluorophore-labeled large RNAs prepared by the current method may be used to investigate RNA structure, folding and mechanism by various fluorescence techniques. In addition, F550/570 and F650/670 may replace [γ-32P]ATP to prepare 5′ labeled RNA for RNA structural and functional investigation, thereby eliminating the need for the unstable and radio-hazardous [γ-32P]ATP

Synthesis of Adenosine Derivatives as Transcription Initiators and Preparation of 5 \u27 Fluorescein- and Biotin-Labeled RNA through One-Step in Vitro Transcription

Expanding our previous finding of an adenosine-initiated transcription system, we now demonstrate that either the 5\u27 site or the N6 site of adenosine nucleotides can be modified extensively without abolishing their ability to initiate transcription under the T7 phi2.5 promoter. Two series of amino derivatives of adenosine nucleotides were synthesized. Fluorescein and biotin groups were coupled to AMP derivatives through linkers of different sizes and hydrophobicities. Both fluorescein- and biotin-conjugated (at either the 5\u27 or N6 site) adenosine nucleotides can act as efficient transcription initiators, producing fluorescein- and biotin-labeled RNA at the specific 5\u27 end by a one-step transcription procedure, eliminating posttranscriptional modification. Furthermore, N6-modified adenosine derivative-initiated transcription synthesizes 5\u27 end modified RNA with a free phosphate group, providing the possibility for further derivatization. The current finding makes easily available a variety of site-specifically functionalized RNA, which may be used in nucleic acid detection, RNA structural and functional investigation, and generation and isolation of novel functional RNA

Instrumentation and Metrology for Single RNA Counting in Biological Complexes or Nanoparticles by a Single-Molecule Dual-View System

Limited by the spatial resolution of optical microscopy, direct detection or counting of single components in biological complexes or nanoparticles is challenging, especially for RNA, which is conformationally versatile and structurally flexible. We report here the assembly of a customized single-molecule dual-viewing total internal reflection fluorescence imaging system for direct counting of RNA building blocks. The RNA molecules were labeled with a single fluorophore by in vitro transcription in the presence of a fluorescent AMP. Precise calculation of identical or mixed pRNA building blocks of one, two, three, or six copies within the bacteriophage phi29 DNA packaging motor or other complexes was demonstrated by applying a photobleaching assay and evaluated by binomial distribution. The dual-viewing system for excitation and recording at different wavelengths simultaneously will enable the differentiation of different complexes with different labels or relative motion of each labeled component in motion machines

NF‐κB, But Not p38 MAP Kinase, is Required for TNF‐α‐Induced Expression of Cell Adhesion Molecules In Endothelial Cells

In response to inflammation stimuli, tumor necrosis factor‐α (TNF‐α) induces expression of cell adhesion molecules (CAMs) in endothelial cells (ECs). Studies have suggested that the nuclear factor‐κB (NF‐κB) and the p38 MAP kinase (p38) signaling pathways play central roles in this process, but conflicting results have been reported. The objective of this study is to determine the relative contributions of the two pathways to the effect of TNF‐α. Our initial data indicated that blockade of p38 activity by chemical inhibitor SB203580 (SB) at 10 µM moderately inhibited TNF‐α‐induced expression of three types of CAMs; ICAM‐1, VCAM‐1 and E‐selectin, indicating that p38 may be involved in the process. However, subsequent analysis revealed that neither 1 µM SB that could completely inhibit p38 nor specific knockdown of p38α and p38β with small interference RNA (siRNA) had an apparent effect, indicating that p38 activity is not essential for TNF‐α‐induced CAMs. The most definitive evidence to support this conclusion was from the experiments using cells differentiated from p38α knockout embryonic stem cells. We could show that deletion of p38α gene did not affect TNF‐α‐induced ICAM‐1 and VCAM‐1 expression when compared with wild‐type cells. We further demonstrated that inhibition of NF‐κB completely blocked TNF‐α‐induced expression of ICAM‐1, VCAM‐1 and E‐selectin. Taken together, our results clearly demonstrate that NF‐κB, but not p38, is critical for TNF‐α‐induced CAM expression. The inhibition of SB at 10 µM on TNF‐α‐induced ICAM‐1, VCAM‐1 and E‐selectin is likely due to the nonspecific effect of SB. J. Cell. Biochem. 105: 477–486, 2008. © 2008 Wiley‐Liss, Inc

Single Protein Encapsulated Doxorubicin as an Efficacious Anticancer Therapeutic

Small‐molecule chemotherapeutics are potent and effective against a variety of malignancies, but common and severe side effects restrict their clinical applications. Nanomedicine approaches represent a major focus for improving chemotherapy, but have met limited success. To overcome the limitations of chemotherapy drugs, a novel single protein encapsulation (SPE)‐based drug formulation and delivery platform is developed and its utility in improving doxorubicin (DOX) treatment is tested. Using this methodology, a series of SPEDOX complexes are generated by encapsulating various numbers of DOX molecules into a single human serum albumin (HSA) molecule. UV/fluorescence spectroscopy, membrane dialysis, and dynamic light scattering techniques show that SPEDOXs are stable and uniform as monomeric HSA and display unique properties distinct from those of DOX and DOX‐HSA mixture. Furthermore, detailed procedures to precisely monitor and control both DOX payload and binding strength to HSA are established. Breast cancer xenograft tumor studies reveal that SPEDOX‐6 treatment displays improved pharmacokinetic profiles, higher antitumor efficacy, and lower DOX accumulation in the heart tissue compared with unformulated DOX. This SPE technology, which does not involve nanoparticle assembly and modifications to either small‐molecule drugs or HSA, may open up a new avenue for developing new drug delivery systems to improve anticancer therapeutics

Loss of Glycosaminoglycan Receptor Binding After Mosquito Cell Passage Reduces Chikungunya Virus Infectivity

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that can cause fever and chronic arthritis in humans. CHIKV that is generated in mosquito or mammalian cells differs in glycosylation patterns of viral proteins, which may affect its replication and virulence. Herein, we compare replication, pathogenicity, and receptor binding of CHIKV generated in Vero cells (mammal) or C6/36 cells (mosquito) through a single passage. We demonstrate that mosquito cell-derived CHIKV (CHIKVmos) has slower replication than mammalian cell-derived CHIKV (CHIKVvero), when tested in both human and murine cell lines. Consistent with this, CHIKVmos infection in both cell lines produce less cytopathic effects and reduced antiviral responses. In addition, infection in mice show that CHIKVmos produces a lower level of viremia and less severe footpad swelling when compared with CHIKVvero. Interestingly, CHIKVmos has impaired ability to bind to glycosaminoglycan (GAG) receptors on mammalian cells. However, sequencing analysis shows that this impairment is not due to a mutation in the CHIKV E2 gene, which encodes for the viral receptor binding protein. Moreover, CHIKVmos progenies can regain GAG receptor binding capability and can replicate similarly to CHIKVvero after a single passage in mammalian cells. Furthermore, CHIKVvero and CHIKVmos no longer differ in replication when N-glycosylation of viral proteins was inhibited by growing these viruses in the presence of tunicamycin. Collectively, these results suggest that N-glycosylation of viral proteins within mosquito cells can result in loss of GAG receptor binding capability of CHIKV and reduction of its infectivity in mammalian cells

Dicer Represses the Interferon Response and the Double-Stranded RNA-Activated Protein Kinase Pathway In Mouse Embryonic Stem Cells

Recent studies have demonstrated that embryonic stem cells (ESCs) are deficient in expressing type I interferons (IFN), the cytokines that play key roles in antiviral responses. However, the underlying molecular mechanisms and biological implications of this finding are poorly understood. In this study, we developed a synthetic RNA-based assay that can simultaneously assess multiple forms of antiviral responses. Dicer is an enzyme essential for RNA interference (RNAi), which is used as a major antiviral mechanism in invertebrates. RNAi activity is detected in wild-type ESCs but is abolished in Dicer knockout ESCs (D-/-ESCs) as expected. Surprisingly, D-/-ESCs have gained the ability to express IFN, which is otherwise deficient in wild-type ESCs. Furthermore, D-/-ESCs have constitutively active double-stranded RNA (dsRNA)-activated protein kinase (PKR), an enzyme that is also involved in antiviral response. D-/-ESCs show increased sensitivity to the cytotoxicity resulting from RNA transfection. The effects of dsRNA can be partly replicated with a synthetic B2RNA corresponding to the retrotransposon B2 short interspersed nuclear element. B2RNA has secondary structure features of dsRNA and accumulates in D-/-ESCs, suggesting that B2RNA could be a cellular RNA that activates PKR and contributes to the decreased cell proliferation and viability of D-/-ESCs. Treatment of D-/-ESCs with a PKR inhibitor and IFNβ-neutralizing antibodies increased cell proliferation rate and cell viability. Based on these findings, we propose that, in ESCs, Dicer acts as a repressor of antiviral responses and plays a key role in the maintenance of proliferation, viability, and pluripotency of ESCs