Halogenated Bases Design and Protocols

Halogenated Bases Design / Protocol

Incorporating halogenated bases into oligonucleotides slated for X-ray crystallography-based structural studies is a convenient method for dealing with the “phase problem” in crystal studies. Light waves (such as X-rays) have both an amplitude (related to intensity) and a phase. When light is physically measured by a suitable detector (photomultiplier tube, CCD camera, etc), the intensity of the light is measured, but the phase information is lost. However, because X-ray crystallography is a diffraction-based method, important information related to the position of the atoms in the crystal is lost. Phase information must be restored for correct structure determination. The classical method for obtaining this phase information is by Multiple Isomorphous Replacement (MIR). For this method, a set of multiple-heavy-atom isomorphic derivatives of the original molecule (ex: a tRNA molecule) are synthesized. Synthesis typically involves soaking a crystal of the native molecule in a heavy atom solution, or co-crystallization with the heavy atom. Each derivative must then be separately crystallized, and X-ray diffraction data collected on it, as is corresponding data from the native molecule. A Patterson difference map is then constructed from all the data in order to reveal the location of the heavy atom(s) in the unit cell. Knowing this location allows both the amplitude and phase of the atoms in the molecule to be determined, which leads to an accurate structure.

While MIR works well, its chief drawback is that many heavy-atom derivatives have to be synthesized, purified and analyzed, making it a complex, time-consuming, trial-by-error method. For nucleic acids, the incorporation of halogenated bases into DNA or RNA (either by chemical or enzymatic synthesis) allows for the use of the alternative method known as Multiwavelength Anomalous Dispersion (MAD). For this method, only one heavy-atom-derivitized DNA/RNA molecule needs to be synthesized, here by targeted substitution of specific bases with their halogenated counterpart (ex: dU with 5-I-dU). All of the diffraction data necessary for correct structural determination is collected from this one sample. The simplicity and speed of this approach makes it the preferred method for structural studies involving DNA, RNA, or protein-nucleic acid complexes.

An excellent introduction to X-ray crystallography of macromolecules is found here.

Halogenated nucleotides are UV-photo-labile molecules. Oligonucleotides containing a halogenated base at a specific position can be UV-crosslinked with another molecule at that location. Halogen-base-modified oligos (especially those with 5-halogenated uracil or 5-halogenated cytosine) are particularly useful in investigational studies into the points of contact (binding locations) between a protein and a nucleic acid in a complex formed between the two (see DNA Protein Crosslinks).

References

(1) Hendrickson, W.; Ogata, C. Phase determination from multiwavelength anomalous diffraction measurements. Meth. Enzymol.. (1997), 276: 494-523.
(2) Herbert, A.G.; Rich, A. A method to identify and characterize Z-DNA binding proteins using a linear oligodeoxynucleotide. Nucleic Acids Res. (1993), 21: 2669-2672.
(3) Schneider, D.J.; Wilcox, S.K.; Zichi, D.; Nieuwlandt, D.; Carter, J.; Gold, L. Improved SELEX and Photo-SELEX. (2008), PCT/US2008/070371 (WO/2009/012410).
(4) Walsh M.A.; Evans G.; Sanishvili R.; Dementieva I.; Joachimiak, A. MAD data collection - current trends. Acta Cryst. (1999), D55: 1726-1732.