Solid-phase methods for sequencing nucleic acids. VII. Chemical degradation of synthetic DNA-RNA hybrid fragments using CCS and DE 81 anion-exchange papers

Synthetic oligo(ribo-deoxyribo)nucleotides were analyzed and characterized by different solid-phase chemical degradation procedures, 5'- and 3'-end labelled mixed fragments were degraded by a slightly modified DNA cleavage procedure using 1 and 10% piperidine for the chain scission reaction and CCS anion-exchange paper. Besides the normal degradation products obtained by the usual modification and strand cleavage reactions of both deoxy- and ribonucleotide residues, additional bands were identified in the sequence patterns resulting from the hydrolysis of the RNA moiety induced by piperidine. Since both degradation reactions cleave the backbone of the mixed DNA-RNA fragments differently and produce nucleotide components with different charges, the degradation products do not interfere and can be resolved by gel electrophoresis on polyacrylamide. In addition, 3'-end labelled DNA-RNA oligomers were degraded by a RNA cleavage procedure using DE 81 anion-exchange paper as solid support. The combination of all three degradation methods allows to confirm the nucleotide sequence.     

Simultaneous sequencing of RNA by solid-phase chemical degradation using DE 81 anion-exchange paper

A solid-phase chemical degradation method for simultaneous sequencing of RNA and RNA fragments has been developed using Whatman DE 81 anion-exchange paper as the support. The approach involves the following operations: (1) immobilization of the 3′-end labeled RNAs or RNA fragments on DE 81 paper; (2) washing; (3) modification reactions; (4) washing; (5) sorting of the paper segments; (6) aniline reaction; (7) lyophilization; (8) desorption of the RNA by TEAB; and (9) lyophilization.     

Solid-phase methods for sequencing of nucleic acids I. Simultaneous sequencing of different oligodeoxyribonucleotides using a new, mechanically stable anion-exchange paper.

A solid-phase method for simultaneous sequencing of large numbers of oligodeoxyribonucleotides has been developed using a new, mechanically stable anion-exchange paper. The excellent mechanical properties of the polymer allow the processing of several paper segments in one reaction vessel or to carry out all necessary operations on a larger area of the paper. In addition, DNA material can be chemically eluted from the new carrier during the piperidine reaction, thus avoiding salt elution of DNA and subsequent ethanol precipitation steps - a prerequisite for sequencing oligonucleotides. The approach involves 7 operations including: i) immobilization; ii) washing; iii) modification; iv) washing; v) sorting of the papers; vi) piperidine reaction and chemical elution and vii) lyophilization. All steps can be carried out in 4 to 5 hours independently of the number of oligonucleotides to be sequenced. It is also possible to sequence small oligonucleotides with 3 to 4 base pairs. The method can be fully automated.     

Optimized conditions for solid-phase sequencing: simultaneous chemical cleavage of a series of long DNA fragments immobilized on CCS anion-exchange paper

A solid-phase method for simultaneous sequencing of ten or more long DNA fragments has been developed, using as support the cellulose matrix for chemical sequencing (CCS), anion-exchange paper [Rosenthal et al., Nucl. Acids Res. 13 (1985) 1173-1184]. We optimized several of the seven steps which include: (i) immobilization; (ii) washing; (iii) modification; (iv) washing; (v) sorting of the paper segments; (vi) piperidine reaction and chemical elution, and (vii) lyophilization. During carrier-supported chemical cleavage with dimethylsulfate (DMS) (G), HCOOH (A + G), KMnO4 (T greater than Pu) and NH2OH (C), losses of immobilized DNA are very low. DNA fragments ranging in length from several hundred bp up to 6 kb can be effectively chemically eluted from CCS paper during the piperidine reaction with an efficiency of more than 90%. Because no DNA salt elution and ethanol precipitation steps are necessary the method is rapid, convenient and allows complete automation.     

New monolith technology for automated anion-exchange purification of nucleic acids

Synthetic nucleic acid analysis often employs pellicular anion-exchange (AE) chromatography because it supports very high efficiency separations while offering means to control secondary structure, retention and resolution by readily modifiable chromatographic conditions. However, these pellicular anion-exchange (pAE) phases do not offer capacity sufficient for lab-scale oligonucleotide (ON) purification. In contrast, monolithic phases produce fast separations at capacities exceeding their pellicular counterparts, but do not exhibit capacities typical of fully porous, bead-based, anion-exchangers. In order to further increase monolith capacity and obtain the selectivity and mass transfer characteristics of pellicular phases, a surface-functionalized monolith was coated with pAE nanobeads (latexes) usually employed on the pellicular DNAPac phase. The nanobead-coated monolith exhibited chromatographic behaviors typical of polymer AE phases. Based on this observation the monolithic substrate surface porosity and latex diameters were co-optimized to produce a hybrid monolith harboring capacity similar to that of fully porous bead-based phases and peak shape approaching that of the pAE phases. We tested the hybrid monolith on a variety of previously developed pAE capabilities including control of ON selectivity, resolution of derivatized ONs, the ability to resolve RNA ONs harboring aberrant linkages at different positions in a single sequence and separation of phosphorothioate diastereoisomers. We compared the yield and purity of an 8 mg ON sample purified on both the new hybrid monolith and a benchmark AE column based on fully porous monodisperse beads. This comparison included an assessment of the relative selectivities of both columns. Finally, we demonstrated the ability to couple AE ON separations with ESI-MS using an automated desalting protocol. This protocol is also useful for preparing ONs for other assays, such as enzyme treatments, that may be sensitive to high salt levels.     

Single-molecule fluorescence methods for the study of nucleic acids.

Single-molecule fluorescence methods and biomechanical tools provide exciting new opportunities to probe biochemical processes in unprecedented detail. The detection and spectroscopy of single fluorophores have recently been used to observe conformational changes and biochemical events involving nucleic acids. A number of fluorescence observables, including localization, quenching, polarization response and fluorescence resonance energy transfer, have been utilized. An exciting new opportunity of combining fluorescence methods and biomechanical tools to study the structural changes and functions of enzymes that participate in nucleic acid metabolism has also arisen.     

Solid-phase synthesis of chemotactic peptides using alpha-azido acids.

Four chemotactic peptides, For-Met-Xxx-Phe-OMe, with an alpha,alpha-disubstituted amino acid at position 2 have been synthesized by the azido acid method [Meldal M, Juliano MA, Jansson AM. 1997. Azido acids in a novel method of solid-phase peptide synthesis. Tetrahedron Lett. 38: 2531-2534] on solid-phase, and were tested for biological activity. Dipropylglycine in the central position (Xxx) was found to be as active as the natural chemotactic peptide for chemotactic activity toward human neutrophils. Higher yields were obtained than previously reported solution-phase syntheses of chemotactic peptides, and EEDQ was used successfully for the difficult solid-phase formylation of amino groups.     

Recent developments in methods for RNA sequencing using in vitro 32P-labeling

A variety of approaches that utilize in vitro 32P-labeling of RNA and of oligonucleotides in the sequence analysis of RNAs are described. These include 1) methods for 5'- and 3'- end labeling of RNAs; 2) end labeling and sequencing of oligonucleotides present in complete T1 RNase or pancreatic RNase digests of RNA; 3) use of random endonucleases, such as nuclease P1, for terminal sequence analysis of end labeled RNAs; and 4) use of base specific enzymes or chemical reagents in the sequence analysis of end-labeled RNAs. Also described is an approach to RNA sequencing, applied so far to tRNAs, which is based on partial and random alkaline cleavage of an RNA to generate a series of overlapping oligonucleotide fragments, all containing the original 3'-end of the RNA. Analysis of the 5'- end group of each of these oligonucleotides (following 5'-end labeling with 32P) provides the sequence of most of the tRNA. The above methods have been used to derive the sequences of several tRNAs, the ribosomal 5S and 5 x 8S RNAs, a viroid RNA, and large segments of both prokaryotic and eukaryotic ribosomal and messenger RNAs.     

Affinity of protein HU for different nucleic acids

The binding of protein HU from Escherichia coli to nucleic acids was investigated by affinity chromatography under various conditions, by a nitrocellulose retention assay and by isopycnic centrifugations in metrizamide gradients. The results indicate that HU has a preference for binding to RNA and single-stranded DNA over double-stranded DNA. The affinity of HU for supercoiled DNA was also less than that of the corresponding relaxed DNA.     

Conformational studies of nucleic acids: III. Empirical multiple correlation functions for nucleic acid torsion angles

There are seven significantly variable torsion angles in each monomer unit of a polynucleotide. Because of this, it is computationally infeasible to consider the energetics of all conformations available to a nucleic acid without the use of simplifications. In this paper, we develop functions suggested by and regression fit to crystallographic data which allow three of these torsion angles, alpha (O3'-P-O5'-C5'), delta (C5'-C4'-C3'-O3') and epsilon (C4'-C3'-O3'-P), to be calculated as dependent variables of those remaining. Using these functions, the seven independent torsions are reduced to four, a reduction in complexity sufficient to allow an examination of the global conformational energetics of a nucleic acid for the remaining independent torsion angles. These functions are the first to quantitatively relate a dependent nucleic acid torsion angle to several different independent angles. In all three cases the data are fit reasonably well, and in one case, alpha, the fit is exceptionally good, lending support for the suitability of the functions in conformational searches. In addition, an examination of the most significant terms in each of the correlation functions allows insight into the physical basis for the correlations.     

Comparison of different antisense strategies in mammalian cells using locked nucleic acids, 2'-O-methyl RNA,phosphorothioates and small interfering RNA

Locked nucleic acids (LNAs) and double-stranded small interfering RNAs (siRNAs) are rather new promising antisense molecules for cell culture and in vivo applications. Here, we compare LNA–DNA–LNA gapmer oligonucleotides and siRNAs with a phosphorothioate and a chimeric 2′-O-methyl RNA–DNA gapmer with respect to their capacities to knock down the expression of the vanilloid receptor subtype 1 (VR1). LNA–DNA–LNA gapmers with four or five LNAs on either side and a central stretch of 10 or 8 DNA monomers in the center were found to be active gapmers that inhibit gene expression. A comparative co-transfection study showed that siRNA is the most potent inhibitor of VR1–green fluorescent protein (GFP) expression. A specific inhibition was observed with an estimated IC₅₀ of 0.06 nM. An LNA gapmer was found to be the most efficient single-stranded antisense oligonucleotide, with an IC₅₀ of 0.4 nM being 175-fold lower than that of commonly used phosphorothioates (IC₅₀ ~70 nM). In contrast, the efficiency of a 2′-O-methyl-modified oligonucleotide (IC₅₀ ~220 nM) was 3-fold lower compared with the phosphorothioate. The high potency of siRNAs and chimeric LNA–DNA oligonucleotides make them valuable candidates for cell culture and in vivo applications targeting the VR1 mRNA.     

Exponential amplification of nucleic acids: new diagnostics using DNA polymerases and RNA replicases

Recent developments in DNA and RNA amplification technology are enabling the design of ultra-sensitive diagnostic assays for infectious diseases. The leading amplification technology is the polymerase chain reaction (PCR). An alternative approach, Q-beta amplification, also promises remarkable speed and precise quantification of assay results.