Gaining target access for deoxyribozymes

Antisense oligonucleotides and ribozymes have been used widely to regulate gene expression by targeting mRNAs in a sequence-specific manner. Long RNAs, however, are highly structured molecules. Thus, up to 90% of putative cleavage sites have been shown to be inaccessible to classical RNA based ribozymes or DNAzymes. Here, we report the use of modified nucleotides to overcome barriers raised by internal structures of the target RNA. In our attempt to cleave a broad range of picornavirus RNAs, we generated a DNAzyme against a highly conserved sequence in the 5' untranslated region (5' UTR). While this DNAzyme was highly efficient against the 5' UTR of the human rhinovirus 14, it failed to cleave the identical target sequence within the RNA of the related coxsackievirus A21 (CAV-21). After introduction of 2'-O-methyl RNA or locked nucleic acid (LNA) monomers into the substrate recognition arms, the DNAzyme degraded the previously inaccessible virus RNA at a high catalytic rate even to completion, indicating that nucleotides with high target affinity were able to compete successfully with internal structures. We then adopted this strategy to two DNAzymes that we had found to be inactive in our earlier experiments. The modified DNAzymes proved to be highly effective against their respective target structures. Our approach may be useful for other ribozyme strategies struggling with accessibility problems, especially when being restricted to unique target sites.     

Universal fluorescent labeling (UFL) method for automated microsatellite analysis.

We have devised a novel method for automated microsatellite analysis using "universal" fluorescent labeling. This system is based on polymerase chain reactions driven by sequence-specific primers and a reporter primer labeled with a fluorescent dye at its 5' end. The forward sequence-specific primer is designed with a tag region bearing no homology to any human genomic sequence. Complementary tag sequences act as templates for the 6-carboxyfluorescein-labeled reporter primer, and those products can be analyzed with an autosequencer. The results we achieved with this assay system were consistent with the results of conventional assays using radioisotope-labeled primers, and diagnosis required less time. Furthermore, the fluorescent-labeled reporter primer is "universal" in that it can be used with different sequence-specific primers designed to carry the appropriate tag sequence at their 5'-ends. Our observations suggest that the "universal" fluorescent labeling method is an efficient tool for analyzing sequence variations in human DNA.     

Development of a hexahistidine-3× FLAG-tandem affinity purification method for endogenous protein complexes in Pichia pastoris

We developed a method for efficient chromosome tagging in Pichia pastoris, using a useful tandem affinity purification (TAP) tag. The TAP tag, designated and used here as the THF tag, contains a thrombin protease cleavage site for removal of the TAP tag and a hexahistidine sequence (6× His) followed by three copies of the FLAG sequence (3× FLAG) for affinity purification. Using this method, THF-tagged RNA polymerases I, II, and III were successfully purified from P. pastoris. The method also enabled us to purify the tagged RNA polymerase II on a large scale, for its crystallization and preliminary X-ray crystallographic analysis. The method described here will be widely useful for the rapid and large-scale preparation of crystallization grade eukaryotic multi-subunit protein complexes.     

Sequence-specific cleavage of RNA in the absence of divalent metal ions by a DNAzyme incorporating imidazolyl and amino functionalities

Two modified 2′-deoxynucleoside 5′-triphosphates have been used for the in vitro selection of a modified deoxyribozyme (DNAzyme) capable of the sequence-specific cleavage of a 12 nt RNA target in the absence of divalent metal ions. The modified nucleotides, a C5-imidazolyl-modified dUTP and 3-(aminopropynyl)-7-deaza-dATP were used in place of TTP and dATP during the selection and incorporate two extra protein-like functionalities, namely, imidazolyl (histidine analogue) and primary amino (lysine analogue) into the DNAzyme. The functional groups are analogous to the catalytic Lys and His residues employed during the metal-independent cleavage of RNA by the protein enzyme RNaseA. The DNAzyme requires no divalent metal ions or other cofactors for catalysis, remains active at physiological pH and ionic strength and can recognize and cleave a 12 nt RNA substrate with sequence specificity. This is the first example of a functionalized, metal-independent DNAzyme that recognizes and cleaves an all-RNA target in a sequence-specific manner. The selected DNAzyme is two orders of magnitude more efficient in its cleavage of RNA than an unmodified DNAzyme in the absence of metal ions and represents a rate enhancement of 10⁵ compared with the uncatalysed hydrolysis of RNA.     

Expression of a Brassic napus glutamate 1-semialdehyde aminotransferase in Escherichia coli and characterization of the recombinant protein

Glutamate 1-semialdehyde aminotransferase (GSA-AT) is a key regulatory enzyme, which converts glutamate 1-semialdehyde (GSA) to 5-aminolevulinic acid (ALA) in chlorophyll biosynthesis. ALA is the universal precursor for the synthesis of chlorophyll, heme, and other tetrapyrroles. To study the regulation of chlorophyll biosynthesis in Brassica napus, two cDNA clones of GSA-AT were isolated for genetic manipulation. A SalI-XbaI fragment from one of the two cDNA clones of GSA-AT was used for recombinant protein expression by inserting it at the 3' end of a calmodulin-binding-peptide (CBP) tag of the pCaln vector. The CBP tagged recombinant protein, expressed in Escherichia coli, was purified to apparent homogeneity in a one step purification process using a calmodulin affinity column. The purified CBP tagged GSA-AT is biologically active and has a specific activity of 16.6 nmol/min/mg. Cleavage of the CBP tag from the recombinant protein with thrombin resulted in 9.2% loss of specific activity. However, removal of the cleaved CBP tag from the recombinant protein solution resulted in 60% loss of specific activity, suggesting possible interactions between the recombinant protein and the CBP tag. The enzyme activity of the CBP tagless recombinant protein, referred as TR-GSA-AT hereafter, was not affected by the addition of pyridoxamine 5' phosphate (PMP). Addition of glutamate and pyridoxal 5' phosphate (PLP) to the TR-GSA-AT enhanced the enzyme activity by 3-fold and 3.6-fold, respectively. Addition of both glutamate and PLP increased the enzyme activity by 4.6-fold. Similar to the GSA-AT of B. napus, the active TR-GSA-AT is a dimeric protein of 88 kDa with 45.5 kDa subunits. As the SalI-XbaI fragment encodes a biologically active GSA-AT that has the same molecular mass as the native GSA-AT, it is concluded that the SalI-XbaI fragment is the coding sequence of GSA-AT. The highly active polyclonal antibodies generated from TR-GSA-AT were used for the detection of GSA-AT of B. napus.     

增强型绿色荧光蛋白的真核表达和纯化

根据编码增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)的开放读码框(open reading frame,ORF)设计引物,PCR方法扩增出5'端带His标签的EGF PORF,利用杆状病毒表达系统构建表达EGFP基因的重组杆状病毒DNA分子,转染sf9细胞.取细胞...     

A 3' exonuclease that specifically interacts with the 3' end of histone mRNA

Metazoan histone mRNAs end in a highly conserved stem-loop structure followed by ACCCA. Previous studies have suggested that the stem-loop binding protein (SLBP) is the only protein binding this region. Using RNA affinity purification, we identified a second protein, designated 3'hExo, that contains a SAP and a 3' exonuclease domain and binds the same sequence. Strikingly, 3'hExo can bind the stem-loop region both separately and simultaneously with SLBP. Binding of 3'hExo requires the terminal ACCCA, whereas binding of SLBP requires the 5' side of the stem-loop region. Recombinant 3'hExo degrades RNA substrates in a 3'-5' direction and has the highest activity toward the wild-type histone mRNA. Binding of SLBP to the stem-loop at the 3' end of RNA prevents its degradation by 3'hExo. These features make 3'hExo a primary candidate for the exonuclease that initiates rapid decay of histone mRNA upon completion and/or inhibition of DNA replication.     

Improved native affinity purification of RNA

RNA biochemical or structural studies often require an RNA sample that is chemically pure, and most protocols for its in vitro production use denaturing polyacrylamide gel electrophoresis to achieve this. Unfortunately, many RNAs do not quantitatively refold into an active conformation after denaturation, creating significant problems for downstream characterization or use. In addition, this traditional purification method is not amenable to studies demanding high-throughput RNA production. Recently, we presented the first general method for producing almost any RNA sequence that employs an affinity tag that is removed during the purification process. Because technical difficulties prevented application of this method to many RNAs, we have developed an improved version that utilizes a different activatable ribozyme and affinity tag that are considerably more robust, rapid, and broadly applicable.     

Purification of plasmids by triplex affinity interaction.

Production of pharmaceutical grade plasmid DNA is an important issue in gene therapy. We developed a method for affinity purification of plasmids by triple helix interaction. This method is based on sequence-specific binding of an oligonucleotide immobilized on a large pore chromatography support to a target sequence on the plasmid. Using design criteria derived from thermodynamic data, we produced a 15mer target sequence which binds strongly to the affinity support under mildly acidic conditions. Plasmid DNA was purified from clarified Escherichia coli lysate by incubation with the affinity beads at pH 5.0 and high NaCl concentration. After extensive washing of the beads, purified plasmid DNA was eluted with alkaline buffer. The purified plasmid showed no RNA or cell DNA contamination in HPLC analysis and total protein concentration was reduced considerably. Due to its mechanical stability and porosity this support can be used in a continuous affinity purification process, which has a high potential for scale up.     

Characterization of 3'hExo, a 3' exonuclease specifically interacting with the 3' end of histone mRNA

The 3' end of mammalian histone mRNAs consisting of a conserved stem-loop and a terminal ACCCA interacts with a recently identified human 3' exonuclease designated 3'hExo. The sequence-specific interaction suggests that 3'hExo may participate in the degradation of histone mRNAs. ERI-1, a Caenorhabditis elegans homologue of 3'hExo, has been implicated in degradation of small interfering RNAs. We introduced a number of mutations to 3'hExo to identify residues required for RNA binding and catalysis. To assure that the introduced mutations specifically target one of these two activities of 3'hExo rather than cause global structural defects, the mutant proteins were tested in parallel for the ability both to bind the stem-loop RNA and to degrade RNA substrates. Our analysis confirms that 3'hExo is a member of the DEDDh family of 3' exonucleases. Specific binding to the RNA requires the SAP domain and two lysines located immediately to its C terminus. 3'hExo binds with the highest affinity to the wild-type 3' end of histone mRNA, and any changes to this sequence reduce efficiency of binding. 3'hExo has only residual, if any, 3' exonuclease activity on DNA substrates and localizes mostly to the cytoplasm, suggesting that in vivo it performs exclusively RNA-specific functions. Efficient degradation of RNA substrates by 3'hExo requires 2' and 3' hydroxyl groups at the last nucleotide. 3'hExo removes 3' overhangs of small interfering RNAs, whereas the double-stranded region is resistant to the enzymatic activity.     

StreptoTag: a novel method for the isolation of RNA-binding proteins

We describe a fast and simple one-step affinity-purification method for the isolation of specific RNA-binding proteins. An in vitro-transcribed hybrid RNA consisting of an aptamer sequence with high binding specificity to the antibiotic streptomycin and a putative protein-binding RNA sequence is incubated with crude extract. After complex formation, the sample is applied to an affinity column containing streptomycin immobilized to Sepharose. The binding of the in vitro-assembled RNA-protein complex to streptomycin-Sepharose is mediated by the aptamer RNA and the specifically bound proteins are recovered from the affinity matrix by elution with the antibiotic. Employing two well-characterized RNA-protein interactions, we tested the performance of this new method. The spliceosomal U1A protein and the bacteriophage MS2 coat protein could be isolated via their appropriate RNA motif containing hybrid RNA from crude yeast extracts in high yield and purity after only one round of affinity purification. As the purification principle is independent of the extract source, this new affinity chromatography strategy that makes use of an in vitro-selected antibiotic-binding RNA as a tag, "StreptoTag," should be applicable to extracts from other organisms as well. Therefore, we propose StreptoTag to be a versatile tool for the isolation of unknown RNA-binding proteins.     

Sequence-specific DNA primer effects on telomerase polymerization activity.

The ribonucleoprotein enzyme telomerase synthesizes one strand of telomeric DNA by copying a template sequence within the RNA moiety of the enzyme. Kinetic studies of this polymerization reaction were used to analyze the mechanism and properties of the telomerase from Tetrahymena thermophila. This enzyme synthesizes TTGGGG repeats, the telomeric DNA sequence of this species, by elongating a DNA primer whose 3' end base pairs with the template-forming domain of the RNA. The enzyme was found to act nonprocessively with short (10- to 12-nucleotide) primers but to become processive as TTGGGG repeats were added. Variation of the 5' sequences of short primers with a common 3' end identified sequence-specific effects which are distinct from those involving base pairing of the 3' end of the primer with the RNA template and which can markedly induce enzyme activity by increasing the catalytic rate of the telomerase polymerization reaction. These results identify an additional mechanistic basis for telomere and DNA end recognition by telomerase in vivo.     

Purification of a nuclear trans-acting factor involved in the regulated transcription of a human immunoglobulin heavy chain gene

The expression of the rearranged human immunoglobulin gamma 1 heavy chain gene (HIG1) was shown to be induced through its enhancer by the positive regulatory trans-acting factor(s) that was contained only in cells of B lineage. The trans-acting factors were purified from mouse myeloma NS1 cells, and HIG1-inducing activity was found mainly in fractions of molecular weight 53-127 kd and in a fraction eluted from a heparin-Sepharose column with 0.5 M KCI. This semipurified fraction contained proteins binding to the conserved octamer sequence, ATGCAAAT, in the promoter region, as well as to sequences in the enhancer region. The 0.5 M KCI eluates from a heparin-Sepharose column were applied to a DNA affinity column of synthetic oligonucleotides of the octamer sequence and the sequence TATTTTAGGAAGCAAA in the HpaII-BgIII region of the HIG1 gene enhancer. The protein eluted from the enhancer sequence-specific DNA affinity column showed a strong inducing activity for the HIG1 gene, and the molecular weight of a predominant protein was 96 kd.     

Recombinant protein purification using complementary peptides as affinity tags

Affinity tags have become highly popular tools for purifying recombinant proteins from crude extracts by affinity chromatography. Besides, short peptides are excellent ligands for affinity chromatography, as they are not likely to cause an immune response in case of leakage into the product, they are more stable than antibodies to elution and cleaning conditions and they usually have very acceptable selectivity. Hydropathically complementary peptides designed de novo show enough selectivity to be used successfully as peptide ligands for protein purification from crude extracts. Recognition specificity and selectivity in the interaction between the complementary peptide pair His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu and Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe have been demonstrated by other authors. In this work, we designed a recombinant protein purification method using a peptide affinity tag that binds to a peptide-binding partner immobilized on a chromatographic matrix. The enhanced green fluorescent protein expressed (EGFP) in Escherichia coli was used as the model. The peptide Gly-Gly-Gly-His-Leu-Leu-Phe-Pro-Ile-Ile-Ile-Ala-Ala-Ser-Leu was synthesized by solid phase using the Fmoc chemistry and immobilized in NHS-Sepharose (PC-Sepharose). Gly residues were added as a spacer arm at the N terminus. The EGFP was expressed either with the fusion tag Lys-Asn-Tyr-Pro-Lys-Lys-Lys-Met-Glu-Lys-Arg-Phe on the C terminus (EGFP-CPTag) or without any fusion tag. After cell disruption, the extract was directly applied to the PC-Sepharose column equilibrated with 20mM sodium phosphate buffer, pH 7.0. The adsorbed EGFP-CPTag was then eluted with 1M Tris. The yield was 98% and the purification factor 4.6. By contrast, EGFP without tag pass through without interacting with the PC-Sepharose column. The method designed can be applied for the purification of other recombinant proteins.