Hybridization of nucleic acids directly in agarose gels

Nucleic acids, both DNA and RNA, separated on agarose gels can be visualized by direct hybridization of the dried gel with appropriate radioactive probes. This method does not involve the transfer of the nucleic acid from the gel. The method requires less manipulation than other procedures; it is extremely rapid, sensitive, and inexpensive. These attributes make this procedure a valuable alternative or supplement to the commonly used methods for visualization by hybridization of nucleic acids separated on agarose gels.     

A rapid and efficient method to remove labelled molecular probes from nucleic acids immobilized on solid supports

A rapid and efficient method is described for the removal of radio-active molecular probes from nucleic acids immobilized on nylon membranes. This method involves boiling in distilled water in a microwave oven. This procedure can be completed within ten minutes, does not require the use of any buffers or reagents, and produces results comparable with conventional buffer-wash procedures recommended by the suppliers of the transfer membranes.     

Precipitation of nucleic acids with poly(ethyleneimine).

Removal of nucleic acids from cell extracts is a common early step in downstream processing for protein recovery. We report on the precipitation of nucleic acids from a homogenate of Saccharomyces cerevisiae by addition of the cationic polyelectrolyte poly(ethyleneimine) (PEI), focusing on the effect of PEI dosage on particle size, protein loss, and extent of nucleic acid removal in both batch and continuous mode. Better than 95% removal of nucleic acids from yeast homogenates was achieved by means of precipitation with PEI with protein losses of approximately 15% with or without previous removal of cell debris. The coprecipitated protein is predominately large molecular weight material and exhibits both low and high isoelectric points. Such treatment does not aggregate the cell debris; size distribution of the precipitated particles from a continuous precipitator is very similar to that for protein precipitation.     

The effects of abscisic Acid on growth and nucleic Acid synthesis in excised embryonic bean axes

Abscisic acid (ABA) is an effective inhibitor of cell elongation in excised embryonic bean axes whether added prior to or after the initiation of cell elongation. Zeatin partially reverses this growth inhibition. ABA inhibits ³²P incorporation into ribosomal RNA, transfer RNA, and DNA but not into the tenaciously bound fraction of elongating axes in a manner resembling 5-fluorouracil, a compound which does not inhibit axis growth. The methylated albumin on kie-selguhr elution profiles of nucleic acids obtained from axes treated with either ABA, 5-fluorouracil, or a combination of the two are similar, and zeatin treatment has little apparent effect on these results. Our results suggest that the inhibition of growth in the axes by ABA is not due to its inhibition of DNA synthesis.     

Nucleic acid quantification by delta pH measurement

Nucleic acids are quantitated by UV absorbance measurement, fluorimetry, or hybridization. While the latter method is time-consuming and requires exact knowledge of the sequence, spectroscopic methods require that the sample does not contain UV-absorbing or fluorescent material. An enzymatic method is the measurement of the hyperchromic change upon cleavage of the nucleic acids by nucleases (Kunitz assay). A variation of this assay makes use of the acidification of the solution upon cleavage. We demonstrate here that microgram nucleic acid quantities can be determined when one employs highly active nonspecific nucleases in conjunction with an instrumental setup consisting of a temperature-controlled mixing chamber and miniaturized pH electrodes. Because this method determines the total amount of phosphodiester bonds cleaved, it is independent of the composition or the secondary structure of the nucleic acid and, under certain precautions, represents a simple and robust alternative to optical assays for the determination of either the total nucleic acid concentration or the activity of nucleases in biochemical samples.     

Fluorescence energy transfer monitored competitive equilibria of nucleic acids: applications in thermodynamics and screening

Precise thermodynamic characterization of nucleic acid complex stability is required to understand a variety of biologically significant events as well as to exploit the specific recognition capabilities of nucleic acids in biotechnology, diagnostics, and therapeutics. The development of a database of nucleic acid thermodynamics with sufficient precision to foster further developments in these areas requires new and improved measurement techniques. The combination of a competitive equilibrium titration with fluorescence energy transfer based detection provides a method for precise measurement of differences in free energy values for nucleic acid duplexes that far exceeds in precision those accessible via conventional methods. The method can be applied to detect and to characterize any deviation in a nucleic acid that alters duplex stability. Such deviations include, but are not limited to, mismatches; single nucleotide polymorphisms (SNP); chemically modified nucleotide bases, sugars or phosphates; and conformational anomalies or folding motifs, such as, loops or hairpins.     

Thermophoretic melting curves quantify the conformation and stability of RNA and DNA

Measuring parameters such as stability and conformation of biomolecules, especially of nucleic acids, is important in the field of biology, medical diagnostics and biotechnology. We present a thermophoretic method to analyse the conformation and thermal stability of nucleic acids. It relies on the directed movement of molecules in a temperature gradient that depends on surface characteristics of the molecule, such as size, charge and hydrophobicity. By measuring thermophoresis of nucleic acids over temperature, we find clear melting transitions and resolve intermediate conformational states. These intermediate states are indicated by an additional peak in the thermophoretic signal preceding most melting transitions. We analysed single nucleotide polymorphisms, DNA modifications, conformational states of DNA hairpins and microRNA duplexes. The method is validated successfully against calculated melting temperatures and UV absorbance measurements. Interestingly, the methylation of DNA is detected by the thermophoretic amplitude even if it does not affect the melting temperature. In the described setup, thermophoresis is measured all-optical in a simple setup using a reproducible capillary format with only 250 nl probe consumption. The thermophoretic analysis of nucleic acids shows the technique's versatility for the investigation of nucleic acids relevant in cellular processes like RNA interference or gene silencing.     

Domains required for nucleic acid binding activities in chloroplast ribonucleoproteins.

Five ribonucleoproteins (or RNA-binding proteins) from tobacco chloroplasts have been identified to date; each of these contains an acidic N-terminal domain (24-64 amino acids) and two conserved RNA-binding domains (82-83 amino acids). All five ribonucleoproteins can bind to ssDNA and dsDNA but show high specificity for poly(G) and poly(U). Here we present the nucleic acid binding activity of each domain using a series of deletion mutant proteins made in vitro from the chloroplast 29 kDa ribonucleoproteins. The acidic domain does not have a positive effect on binding activities and proteins lacking this domain show higher affinities for nucleic acids than the wild-type proteins. Mutant proteins containing single RNA-binding domains can bind to poly(G) and poly(U), though with lower affinities than proteins containing two RNA-binding domains. The spacer region (11-37 amino acids) between the two RNA-binding domains does not interact with poly(G) or poly(U) by itself, but is required for the additive activity of the two RNA-binding domains. Proteins consisting of two RNA-binding domains but lacking the spacer have the same activity as those containing only one RNA-binding domain. Possible roles for each domain in chloroplast ribonucleoproteins are discussed.     

The interaction of adriamycin and adriamycin analogues with nucleic acids in the B and A conformations.

The reinforced intercalative binding to DNA typical of adriamycin and daunomycin can still occur if there is epimerisation at C4' or if the O-methyl group is lost or if the 9-substituents are deleted or if the 4'-hydroxyl group is lost. In the latter two cases however, there is a reduction in affinity for the DNA, supporting the suggested role of the 9-hydroxyl and 4'-hydroxyl groups in secondary stabilization of the complex. Epimerisation at C-1' or at C-3' alters but does not abolish the intercalative mode of binding to DNA whereas epimerisation at C-7 precludes intercalation of the chromophore into the helix of DNA. In contrast to the interaction with the B-form found in DNA, the parent drugs do not intercalate into nucleic acids possessing the A-conformation and none of the above-mentioned structural changes will allow intercalation into A-form nucleic acids.     

Nucleic acid binding properties of the nucleic acid chaperone domain of hepatitis delta antigen

The N terminal region of hepatitis delta antigen (HDAg), referred to here as NdAg, has a nucleic acid chaperone activity that modulates the ribozyme activity of hepatitis delta virus (HDV) RNA and stimulates hammerhead ribozyme catalysis. We characterized the nucleic acid binding properties of NdAg, identified the structural and sequence domains important for nucleic acid binding, and studied the correlation between the nucleic acid binding ability and the nucleic acid chaperone activity. NdAg does not recognize the catalytic core of HDV ribozyme specifically. Instead, NdAg interacts with a variety of nucleic acids and has higher affinities to longer nucleic acids. The studies with RNA homopolymers reveal that the binding site size of NdAg is around nine nucleotides long. The extreme N terminal portion of NdAg, the following coiled-coil domain and the basic amino acid clusters in these regions are important for nucleic acid binding. The nucleic acid–NdAg complex is stabilized largely by electrostatic interactions. The formation of RNA–protein complex appears to be a prerequisite for facilitating hammerhead ribozyme catalysis of NdAg and its derivatives. Mutations that reduce the RNA binding activity or high ionic strength that destabilizes the RNA–protein complex, reduce the nucleic acid chaperone activity of NdAg.     

A simple and sensitive method for detection of nucleic acids fixed on nylon-based filters

Two methods for detection of nucleic acids, fixed on nylon membranes, are described. A negative staining, using the Auro-dye technique described for proteins, results in a sensitive and fast detection of nucleic acids fixed on nylon membranes, which are routinely used in molecular biology. Subsequent hybridization treatment does not influence the staining. A positive staining with a cationic iron colloid is also described. Sensitivity and application of both methods are discussed.     

Spectroscopic properties of ethidium monoazide: a fluorescent photoaffinity label for nucleic acids.

The non-covalent binding of ethidium monoazide to nucleic acids is entirely analogous to that of ethidium (binding constant approximately 2-3 X 10(5) M). The ethidium monoazide can be photochemically covalently linked to nucleic acids in high yield, up to 75%, by long wavelength light. The fluorescence of ethidium monoazide and ethidium crosslinked to nucleic acids show the same environmental sensitivity as does the fluorescence of ethidium. These properties of ethidium monoazide indicate its use as a fluorescent photoaffinity label for nucleic acids. Ethidium diazide can be photochemically linked to nucleic acids but appears to have properties substantially different from those of ethidium.     

In vitro selection of nucleoprotein enzymes.

Natural nucleic acids frequently rely on proteins for stabilization or catalytic activity. In contrast, nucleic acids selected in vitro can catalyze a wide range of reactions even in the absence of proteins. To augment selected nucleic acids with protein functionalities, we have developed a technique for the selection of protein-dependent ribozyme ligases. After randomizing a previously selected ribozyme ligase, L1, we selected variants that required one of two protein cofactors, a tyrosyl transfer RNA (tRNA) synthetase (Cyt18) or hen egg white lysozyme. The resulting nucleoprotein enzymes were activated several thousand fold by their cognate protein effectors, and could specifically recognize the structures of the native proteins. Protein-dependent ribozymes can potentially be adapted to novel assays for detecting target proteins, and the selection method's generality may allow the high-throughput identification of ribozymes capable of recognizing a sizable fraction of a proteome.     

Semi-dry electroblotting of DNA and RNA from agarose and polyacrylamide gels.

Semi-dry electroblotting of nucleic acids was originally of interest for its speed and convenience, but it had significant limitations in reliability of transfer. This work describes optimalization of the buffer systems and general conditions for use that enable the semi-dry method of nucleic acids transfer to yield quantitative, fast and reliable results.     

Fluorescence energy transfer monitored competitive equilibria of nucleic acids: Applications in thermodynamics and screening

Precise thermodynamic characterization of nucleic acid complex stability is required to understand a variety of biologically significant events as well as to exploit the specific recognition capabilities of nucleic acids in biotechnology, diagnostics, and therapeutics. The development of a database of nucleic acid thermodynamics with sufficient precision to foster further developments in these areas requires new and improved measurement techniques. The combination of a competitive equilibrium titration with fluorescence energy transfer based detection provides a method for precise measurement of differences in free energy values for nucleic acid duplexes that far exceeds in precision those accessible via conventional methods. The method can be applied to detect and to characterize any deviation in a nucleic acid that alters duplex stability. Such deviations include, but are not limited to, mismatches; single nucleotide polymorphisms (SNP); chemically modified nucleotide bases, sugars or phosphates; and conformational anomalies or folding motifs, such as, loops or hairpins. © 2002 Wiley Periodicals, Inc. Biopoly (Nucleic Acid Sci) 61: 214–223, 2002     

Adding functional entities to plasmids

Non-viral gene therapy constitutes an alternative to the more common use of viral-mediated gene transfer. Most gene transfer methods using naked DNA are based upon non-sequence-specific interactions between the nucleic acid and cationic lipids (lipoplex) or polymers (polyplex). We have developed a technology in which functional entities hybridize in a sequence-specific manner to the nucleic acid (bioplex). This technology is still in its infancy, but has the potential to become a useful tool, since it allows the construction of highly defined complexes containing a variety of functional entities. In its present form the bioplex technology is based upon the use of peptide/nucleic acids (PNA) as anchors. Single, or multiple, functional entities are directly coupled to the anchors. By designing plasmids, or oligonucleotides, with the corresponding anchor target sequence, complexes with desired composition can easily be generated. The long-term aim is to combine functional entities in order to achieve optimal, synergistic interactions allowing enhanced gene transfer in vivo.     

In vitro stability of alpha-helical peptide nucleic acids (alphaPNAs)

Alpha-helical peptide nucleic acids (alphaPNAs) are synthetic molecules that merge the alpha-helix secondary structure of peptides with the codified Watson-Crick base pairing capability of nucleic acids. It is now demonstrated that alphaPNAs made up of either L- or D-amino acids are resistant to degradation by the proteases present in human serum. The increased stability of alphaPNAs towards proteases may be attributable to the presence of unnatural nucleoamino acid residues [-NHCH(CH(2)OCH(2)B)CO-, where B=thymine or cytosine] since the replacement of these amino acids by serine yields a control peptide that does break down in human serum. The stability of alphaPNAs towards proteases makes them attractive candidates for further development as antisense agents.     

Derivatization of unprotected polynucleotides.

A simple and efficient method for attaching amines to the terminal 5'-phosphate of unprotected oligonucleotides or nucleic acids in aqueous solution is described. The method is applicable to low molecular-weight amines, polypeptides, or proteins. The terminal 5'-phosphate of an oligonucleotide or nucleic acid reacts with a water-soluble carbodiimide in imidazole buffer at pH 6 to give good yields of the 5'-phosphorimidazolide. Exposure of the phosphorimidazolide to amine-containing molecules in aqueous solution results in the production of a wide range of stable phosphoramidates in high yield. The exposure of polynucleotides to carbodiimide does not result in significant breakage of phosphodiester bonds or damage to nucleoside bases. The biological activity of a drug resistant plasmid is not affected. The direct condensation of polynucleotides with amines in 1-methylimidazole buffer is also possible. However, it is not a satisfactory preparative method if the ligand is sensitive to carbodiimide.     

Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators

The highest sensitivity nucleic acid gel stains developed to date are optimally excited using short-wavelength ultraviolet or visible light. This is a disadvantage for laboratories equipped only with 306- or 312-nm UV transilluminators. We have developed a new unsymmetrical cyanine dye that overcomes this problem. This new dye, SYBR Gold nucleic acid gel stain, has two fluorescence excitation maxima when bound to DNA, one centered at approximately 300 nm and one at approximately 495 nm. We found that when used with 300-nm transillumination and Polaroid black-and-white photography, SYBR Gold stain is more sensitive than ethidium bromide, SYBR Green I stain, and SYBR Green II stain for detecting double-stranded DNA, single-stranded DNA, and RNA. SYBR Gold stain's superior sensitivity is due to the high fluorescence quantum yield of the dye-nucleic acid complexes ( approximately 0.7), the dye's large fluorescence enhancement upon binding to nucleic acids ( approximately 1000-fold), and its capacity to more fully penetrate gels than do the SYBR Green gel stains. We found that SYBR Gold stain is as sensitive as silver staining for detecting DNA-with a single-step staining procedure. Finally, we found that staining nucleic acids with SYBR Gold stain does not interfere with subsequent molecular biology protocols.