Spin labeling of oligonucleotides with the nitroxide TPA and use of PELDOR, a pulse EPR method, to measure intramolecular distances

In this protocol, we describe the facile synthesis of the nitroxide spin-label 2,2,5,5-tetramethyl-pyrrolin-1-oxyl-3-acetylene (TPA) and then its coupling to DNA/RNA through Sonogashira cross-coupling during automated solid-phase synthesis. Subsequently, we explain how to perform distance measurements between two such spin-labels on RNA/DNA using the pulsed electron paramagnetic resonance method pulsed electron double resonance (PELDOR). This combination of methods can be used to study global structure elements of oligonucleotides in frozen solution at RNA/DNA amounts of approximately 10 nmol. We especially focus on the Sonogashira cross-coupling step, the advantages of the ACE chemistry together with the appropriate parameters for the RNA synthesizer and on the PELDOR data analysis. This procedure is applicable to RNA/DNA strands of up to approximately 80 bases in length and PELDOR yields reliably spin-spin distances up to approximately 6.5 nm. The synthesis of TPA takes approximately 5 days and spin labeling together with purification approximately 4 days. The PELDOR measurements usually take approximately 16 h and data analysis from an hour up to several days depending on the extent of analysis.     

Use of synthetic oligonucleotides in gene isolation and manipulation

Great progress has occurred in the techniques of synthesis of DNA molecules of defined sequences in terms of speed, length of the obtained oligonucleotides, and automation of the processes. Corresponding progress also occurred in the ways of using synthetic DNA in molecular biology and recombinant DNA research. Screening of cloned DNA sequence banks with long, unique oligonucleotides, provided a new approach to isolate the genes for proteins which are present in very small quantity. This technique can present considerable advantages over the more classical use of mixtures of oligonucleotides, in reducing the number of potentially positive clones on a primary screen, and enabling cloning with a minimum of amino acid sequence data. Synthetic oligonucleotides also provide the basis of a set of techniques for site-directed mutagenesis of DNA sequences. This allows the possibility of engineering the structure of particular proteins, and the properties of new variants can be tested by expressing the protein in a heterologous host. An example of this approach is the production of variants of human alpha 1-antitrypsin. A variant where valine replaces the methionine at the active site is equally active as an antielastase, but no longer susceptible to oxidative inactivation. A second variant, where arginine replaces the methionine, now functions as an antithrombin, but no longer inhibits elastase. Total gene synthesis is now feasible for larger and larger genes, and some of the recent strategies of whole gene synthesis are presented.     

Analysis of thermal melting curves

T(m) is defined as Temperature of melting or, more accurately, as temperature of midtransition. This term is often used for nucleic acids (DNA and RNA, oligonucleotides and polynucleotides). A thermal denaturation experiment determines the stability of the secondary structure of a DNA or RNA and aids in the choice of the sequences for antisense oligomers or PCR primers. Beyond a simple numerical value (the T(m)), a thermal denaturation experiment, in which the folded fraction of a structure is plotted vs. temperature, yields important thermodynamic information. We present the classic problems encountered during these experiments and try to demonstrate that a number of useful pieces of information can be extracted from these experimental curves.     

High throughput parallel synthesis of oligonucleotides with 1536 channel synthesizer

A 1536 channel oligonucleotide synthesizer, the MultiSyn, was developed with the capability to simultaneously synthesize 1536 oligonucleotides of 20mer length in 10 h. The instrument was designed to synthesize different sequences of various lengths in micro-wells and has synthesized oligonucleotides as long as 119 nt with reasonably good yields using CPG beads of 1000 Å pore size. The instrument consists of four 384 channel synthesis modules. Phosphoramidite chemistry was employed and step yields as high as 99.3% were achieved. The enhancement of oligonucleotide synthesis throughput is accomplished by increasing the spatial density of reaction wells. We have identified several parameters that are critical in achieving a good synthesis yield and negligible failure rate in small reaction wells. The coefficient of variation (CV) of product yields in 1536 reaction wells was 20%. The quality of the product was examined by capillary electrophoresis and mass spectrometry. The instrument has robustly synthesized oligonucleotides of various lengths for use as primers and probes for PCR amplifications, oligonucleotide microarrays and genotyping applications. This high throughput oligonucleotide synthesizer is a useful instrument for genomic applications, which require tens of thousands of probes or primers in a short time.     

Interpreting oligonucleotide microarray data to determine RNA secondary structure: application to the 3' end of Bombyx mori R2 RNA

A method to deduce RNA secondary structure on the basis of data from microarrays of 2'-O-methyl RNA 9-mers immobilized in agarose film on glass slides is tested with a 249 nucleotide RNA from the 3' end of the R2 retrotransposon from Bombyx mori. Various algorithms incorporating binding data and free-energy minimization calculations were compared for interpreting the data to provide possible secondary structures. Two different methods give structures with 100 and 87% of the base pairs determined by sequence comparison. In contrast, structures predicted by free-energy minimization alone by Mfold and RNAstructure contain 52 and 72% of the known base pairs, respectively. This combination of high throughput microarray techniques with algorithms using free-energy calculations has potential to allow for fast determination of RNA secondary structure. It should also facilitate the design of antisense and siRNA oligonucleotides.     

Nucleotide sequence study of mouse 5.8S ribosomal RNA.

The primary structure of 5.8S mouse ribosomal RNA has been studied and compared to the structures previously established for other animal species. The results obtained show that mouse 5.8S ribosomal RNA yields pancreatic oligonucleotides with the same nucleotide sequence as the homologous oligonucleotides from rat cells. Furthermore T1 oligonucleotides of 5.8S ribosomal RNA from rat, mouse and human cells behave identically on fingerprinting fractionation and have the same composition as judged by pancreatic digestion. These results strongly suggest that the primary structures of 5.8S ribosomal RNA from rat, mouse and human cells are identical. This identity of structure is also found when the presence of several modified bases (psi and methylated bases) is considered. The findings emphasize the remarkable evolutionary stability of ribosomal gene structure. Comparison of the terminal regional of 5.8S RNA with those of 18S RNA reveals differences which imply a more complex mechanism underlying the maturation of 45S precursor RNA than the finding of identical structure would have suggested.     

Branched RNA

The only RNA molecules known to be branched are circular structures with tails known as lariats that arise during nuclear pre-mRNA splicing. Lariats accumulate within a large multicomponent particle called a spliceosome that forms upon the addition of unspliced mRNA to nuclear extracts. Recently an RNA molecule has been observed to catalyze branch formation. In this case a single intron of a yeast mitochondrial pre-mRNA participates in a self-splicing reaction that results in the accumulation of branched lariats that are processed to correctly spliced exons. An enzyme highly specific for branch removal found in the same extracts that form branches during pre-mRNA splicing can debranch RNA lariats to their linear forms without loss of nucleotides. The chemical synthesis of branched RNA has recently been achieved. High yields of sequence-specific oligonucleotides are now available for the analysis of RNA splicing by techniques dependent on branch-site recognition.     

Synthesis and hybridization properties of inverse oligonucleotides.

The synthesis of adenine and thymine cyclopentylethyl nucleosides is presented. This novel constrained monomeric building block is very difficult to incorporate into oligonucleotides. It was introduced in 13mer oligodeoxynucleotide sequences at a single position using H-phosphonate chemistry. Phosphoramidite chemistry completely failed in this particular case. The H-phosphonate building blocks were obtained starting from the corresponding phosphoramidites. Stability of duplexes with RNA and DNA is significantly reduced.     

Chemical synthesis of the 5'-terminal structure of U1 RNA

A new method for the synthesis of N2,N2-dimethylguanosine (DMG) is developed by reductive C-S bond cleavage by use of tributyltin hydride. An improved method for the synthesis of the key intermediate (1) for construction of the 5'-terminal structure of U1 RNA, which has a trimethylated cap (TMG) structure at its 5' end, is also described. By the use of 1, several TMG-capped ribonucleosides and oligonucleotides were synthesized.     

A fluorimetric assay for available lysine in proteins

A two-dimensional fingerprinting gel system that provides sensitive analyses with high resolution of T1-resistant oligonucleotides of large RNA molecules is described. Unique oligonucleotides less than 30 bases in length are recovered quantitatively while longer oligonucleotides are recovered in very large (～90%) yields by active transfer of the fingerprint to DEAE paper. After elution of the oligonucleotides from DEAE paper, secondary analysis is performed by digestion of oligonucleotides with pancreatic RNase and separation of the products by high-voltage electrophoresis on polyethyleneimine cellulose. The complete analysis of up to 40 oligonucleotides can be accomplished within 4 days.     

A simple,universal, efficient PCR-based gene synthesis method: Sequential OE-PCR gene synthesis

Herein we present a simple, universal, efficient gene synthesis method based on sequential overlap extension polymerase chain reactions (OE-PCRs). This method involves four key steps: (i) the design of paired complementary 54-mer oligonucleotides with 18 bp overlaps, (ii) the utilisation of sequential OE-PCR to synthesise full-length genes, (iii) the cloning and sequencing of four positive T-clones of the synthesised genes and (iv) the resynthesis of target genes by OE-PCR with correct templates. Mispriming and secondary structure were found to be the principal obstacles preventing successful gene synthesis and were easily identified and solved in this method. Compensating for the disadvantages of being laborious and time-consuming, this method has many attractive advantages, such as the ability to guarantee successful gene synthesis in most cases and good allowance for Taq polymerase, oligonucleotides, PCR conditions and a high error rate. Thus, this method provides an alternative tool for individual gene synthesis without strict needs of the high-specialised experience.     

A flexible light-directed DNA chip synthesis gated by deprotection using solution photogenerated acids

Oligonucleotide microarrays or oDNA chips are effective decoding and analytical tools for genomic sequences and are useful for a broad range of applications. Therefore, it is desirable to have synthesis methods of DNA chips that are highly flexible in sequence design and provide high quality and general adoptability. We report herein, DNA microarray synthesis based on a flexible biochip method. Our method simply uses photogenerated acid (PGA) in solution to trigger deprotection of the 5′-OH group in conventional nucleotide phosphoramidite monomers (i.e. PGA-gated deprotection), with the rest of the reactions in the synthesis cycle the same as those used for routine synthesis of oligonucleotides. The complete DNA chip synthesis process is accomplished on a regular DNA synthesizer that is coupled with a UV-VIS projection display unit for performing digital photolithography. Using this method, oDNA chips containing probes of newly discovered genes can be quickly and easily synthesized at high yields in a conventional laboratory setting. Furthermore, the PGA-gated chemistry should be applicable to microarray syntheses of a variety of combinatorial molecules, such as peptides and organic molecules.     

Selenium modification of nucleic acids: preparation of oligonucleotides with incorporated 2'-SeMe-uridine for crystallographic phasing of nucleic acid structures

This protocol describes a method for introducing an anomalously scattering atom into oligonucleotides at the 2'-position of uridine by conventional solid-phase synthesis. The 2'-SeMe ribose modification is particularly attractive for derivatization of RNA to facilitate crystal structure determination. The estimated time for the synthesis and HPLC purification of oligonucleotides with incorporated 2'-SeMe-uridine residues is approximately 46 h for 'trityl on' and approximately 32 h for 'trityl off' methods, respectively.     

Antiproliferative activity of G-quartet-forming oligonucleotides with backbone and sugar modifications

Oligonucleotide-based therapies have considerable potential in cancer, viral, and cardiovascular disease therapies. However, it is becoming clear that the biological effects of oligonucleotides are not solely due to the intended sequence-specific interactions with nucleic acids. Oligonucleotides are also capable of interacting with numerous cellular proteins owing to their polyanionic character or specific secondary structure. We have examined the antiproliferative activity, protein binding, and G-quartet formation of a series of guanosine-rich oligonucleotides, which are analogues of GRO29A, a G-quartet forming, growth-inhibitory oligonucleotide, whose effects we have previously described [Bates P. J., Kahlon, J. B., Thomas, S. D., Trent, J. O., and Miller, D. M. (1999) J. Biol. Chem. 274, 26369-26377]. The GRO29A analogues include phosphorothioate (PS29A), 2'-O-methyl RNA (MR29A), and mixed DNA/2'-O-methyl RNA (MRdG29A) oligonucleotides. We demonstrate by UV spectroscopy that all of the modified analogues form stable structures, which are consistent with G-quartet formation. We find that the phosphorothioate and mixed DNA/2'-O-methyl analogues are able to significantly inhibit proliferation in a number of tumor cell lines, while the 2'-O-methyl RNA has no significant effects. Similar to the original oligonucleotide, GRO29A, the growth inhibitory oligonucleotides were able to compete with the human telomere sequence oligonucleotide for binding to a specific cellular protein. The less active MR29A does not compete significantly for this protein. On the basis of molecular modeling of the oligonucleotide structures, it is likely that the inactivity of MR29A is due to the differences in the groove structure of the quadruplex formed by this oligonucleotide. Interestingly, all GRO29A analogues, including an unmodified DNA phosphodiester oligonucleotide, are remarkably resistant to nuclease degradation in the presence of serum-containing medium, indicating that secondary structure plays an important role in biological stability. The remarkable stability and strong antiproliferative activity of these oligonucleotides confirm their potential as therapeutic agents.     

Versatile derivatisation of solid support media for covalent bonding on DNA-microchips

A chemistry was developed that permits on DNA-arrays both the covalent immobilisation of pre-fabricated nucleic acids-such as oligonucleotides, PCR-products or peptide nucleic acid oligomers-and the in situ synthesis of such compounds on either glass or polypropylene surfaces. Bonding was found to be stable even after some 30 cycles of stripping. Due to a dendrimeric structure of the linker molecule, the loading can be modified in a controlled manner and increased beyond the capacity of glass without negative effects on hybridisation efficiency. Also, the chemistry warrants the modulation of other surface properties such as charge or hydrophobicity. Preferentially, attachment of nucleic acids takes place only via the terminal amino-group of amino-modified oligonucleotides or the terminal hydroxyl-group of unmodified molecules so that the entire molecule is accessible to probe hybridisation. This derivatisation represents a support chemistry versatile enough to serve nearly all current forms of DNA-arrays or microchips.     

Selenium derivatization and crystallization of DNA and RNA oligonucleotides for X-ray crystallography using multiple anomalous dispersion

We report here the solid phase synthesis of RNA and DNA oligonucleotides containing the 2′-selenium functionality for X-ray crystallography using multiwavelength anomalous dispersion. We have synthesized the novel 2′-methylseleno cytidine phosphoramidite and improved the accessibility of the 2′-methylseleno uridine phosphoramidite for the synthesis of many selenium-derivatized DNAs and RNAs in large scales. The yields of coupling these Se-nucleoside phosphoramidites into DNA or RNA oligonucleotides were over 99% when 5-(benzylmercapto)-1H-tetrazole was used as the coupling reagent. The UV melting study of A-form dsDNAs indicated that the 2′-selenium derivatization had no effect on the stability of the duplexes with the 3′-endo sugar pucker. Thus, the stems of functional RNA molecules with the same 3′-endo sugar pucker appear to be the ideal sites for the selenium derivatization with 2′-Se-C and 2′-Se-U. Crystallization of the selenium-derivatized oligonucleotides is also reported here. The results demonstrate that this 2′-selenium functionality is suitable for RNA and A-form DNA derivatization in X-ray crystallography.     

Oligonucleotide charge reversal: 2'-O-lysylaminohexyl modified oligonucleotides

A novel cationic building nucleoside building block designed for antisense and siRNA oligonucleotides is presented. Protected L-lysine was coupled to 2'-O-aminohexyluridine and the resulting nucleoside was phosphitylated for automated oligonucleotide synthesis. An increasing number of these 2'-O-lysylaminohexyl nucleosides lowered the melting temperature of desoxy-thymidine homododecamers, but the decrease was lower than that for DNA/RNA hybrids. Incubation with an exonuclease showed the exceptionally high resistance against enzymatic degradation. CD spectrometry revealed a gradual transition towards an A-type oligonucleotide structure. Based on these data, the cationic building block is particularly suited for gapmer antisense as well as siRNA oligonucleotides.