Analysis of Oligonucleotides

RP-HPLC and ion-exchange chromatography discussed in the previous section are the most important methods for both analysis and purification of ODNs. A variety of protocols and commercial columns has become available based on these methods. 

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Biomolecular MS and in particular MALDI-TOF-MS (see Sections 2.1.22 and 2.2.1) permit the routine analysis of oligonucleotides up to 70-mers, intact nucleic acids, and the direct detection of DNA products with no primer labels with an increase in analysis speed and mass accuracy especially in contrast to traditional DNA separation techniques such as slab gels or capillary electrophoresis. Applications focus on the characterization of single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). Precise and accurate gene expression measurements show relative and absolute numbers of target molecules determined independently of the number of PCR cycles. DNA methylation can be studied quantitatively. 

Hong SP, Kim NK, Hwang SG, Chung HJ, Kim S, et al. 2004. Detection of hepatitis B virus YMDD variants using mass spectrometric analysis of oligonucleotide fragments. J Hepatol 40 837. 

Soper, S.A., Ford, S.M., Xu, Y.C., Qi, S.Z., McWhorter, S., Lassiter, S., Patterson, D., Bmch, R.C., Nanoliter-scale sample preparation methods directly coupled to polymethylmethacrylate-based microchips and gel-filled capillaries for the analysis of oligonucleotides.. /. Chromatogr. A 1999, 853, 107-120. 

Alazard, D., Tran, L., Allen, J. R., Weisburg, W. G., and Russell, J. (1998). Ion chromatography analysis of oligonucleotide mixtures in the medical diagnostics industry. Int. Symp. High Performance Liq. Phase Sep. Related Tech., HPLC 98, 22nd, St. Louis, MO, 1998. 

Apffel, A., Chakel, J. A., Fisher, S., Lichtenwalter, K., and Hancock, W. S. (1997). Analysis of oligonucleotides by HPLC-electrospray ionization mass spectrometry. Anal. Chem. 69, 1320-1325. 

Reviews on the mass spectrometry analysis of oligonucleotides have been published in the last few years. [158-161]... 

A second difficulty in the analysis of oligonucleotides is their easy fragmentation. As will be discussed in more detail, the oligonucleotides tend to lose their nucleic bases by a 1,2-elimination mechanism. Fragmentation of the nucleic chain at either the 5 or 3 side of the sugar having lost a base then can occur. This stability problem is more important for MALDI than for ESI analysis. 

The success of the analysis of oligonucleotides is strongly dependent on the choice of the matrix and the preparation of the sample. A major improvement has resulted from the introduction of new matrices specific for the oligonucleotides. An example is 3-hydroxypicolinic acid. [165] Several strategies for sample preparation have been adopted, with the principal goal of eliminating interference from the ubiquitous alkali metal ions a combination of the use of cation exchange columns to replace the alkali metal ions by ammonium and the addition of some ammonium salt to the sample. 

Paulus A, Ohms JI (1990) Analysis of oligonucleotides by capillary gel electrophoresis. J Chromatogr 507 113-123. 

Briichert, W., Kruger, R., Tholey, A., Montes-Bayon, M., Bettmer, J. A novel approach for analysis of oligonucleotide-cisplatin interactions by continuous elution gel electrophoresis coupled to isotope dilution inductively coupled plasma mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. Electrophoresis 29, 1451-1459 (2008)... 

Guerra CE. Analysis of oligonucleotide microarrays by 3 end labeling using fluorescent nucleotides and terminal transferase. Biotechniques 2006 41 53-56. 

A last problem in nucleotide mass spectrometry is related to the dinucleotides. Hunt and Hignite tried to prepare a matrix of several ribodinucleo-tides (silylated) for further application to the sequential analysis of oligonucleotides. The fact that common enzymatic and/or chemically induced hydrolyses do not stop at the nucleotide level, but rather continue until they become nucleosides, does not reduce the merit of their work. That work, as well as that of Biemann, enable one to identify any dinucleotide pair of the type B,pB2, where p stands for phosphate. This information is of very limited use, however, because from any given RNA, for instance, one obtains practically any possible B1PB2 combination of dinucleotides. 

Horton JD, Shah NA, Warrington JA, Anderson NN, Park SW, Brown MS, Goldstein JL (2003) Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes. Proc Natl Acad Sci US A 100 12027-12032... 

The general principle of mass spectrometry (MS) is to produce, separate and detect gas phase ions. Traditionally, thermal vaporization methods are used to transfer molecules into the gas phase. The classical methods for ionization are electron impact (El) and chemical ionization (Cl). Most biomolecules, however, undergo severe decomposition and fragmentation under the conditions of both methods. Consequently, the capabilities of mass spectrometry have been limited to molecules the size of dinucleotides [1]. Analysis of oligonucleotides with a mass range of up to 3000 Da became feasible with the development of plasma desorption (PD) methods [2]. However, until the invention of soft ionization techniques such as ESI- and MALDI MS, mass spectrometric tools were not widely considered for routine applications in biological sciences. 

Li, G, and Wong, W.H. 2001. Model-based analysis of oligonucleotide arrays Expression index computation and outlier detection. Proc Natl Acad Sci USA 98 31-36. 

A series of reviews has been published on the analysis of oligonucleotides using CE [183-185]. A comprehensive review on separation of mono, oligo- and polymeric nucleic acids by this method has also been published [186], In contrast to RP-HPLC and ion-exchange chromatography, CE - which has been established as the third technique of choice -is a purely analytical method [187]. Ion-exchange HPLC fails particularly with SODNs [185]. RP-HPLC does not at present allow baseline separation of SODNs, which differ by only one base pair [188]. Thus, control of preparative purifications by RP-HPLC has become an important application of CGE. Table 11 illustrates the resolution efficiency of CGE when compared with other methods. 

Engels, W, Schweitzer, M, Analysis of Oligonucleotides, in Antisense - From Technology to Therapy, R. Schliengensiepen, Schliengensiepen, K.-H., Brysch, W., Editor. 1997, Blackwell Science Ltd. Berlin- Vienna, p. 78. 

Pesole G., Gissi C., Grillo G., Licciulli F., Liuni S., Saccone C, (2000). Analysis of oligonucleotide AUG start codon context in eukariotic mRNAs. Gene 261 85-91. 

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