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To learn more about RNA synthesis and PROLIGO recommendations

RNA STRUCTURE

The structure of the RNA is very similar to the DNA structure. Each molecule is a polymer consisting respectively of ribonucleosides or deoxyribonucleosides joined by 3' to 5' phosphodiester bonds. In RNA, one of the four major bases is uracil instead of thymidine (Figure 1)

Figure 1:

RNA Oligos: Figure 1


INTRODUCTION TO RNA SYNTHESIS

The chemistry of RNA synthesis is identical to the DNA synthesis except for the presence of an additional protecting group at the 2' hydroxyl position of ribose. This position is protected with silyl groups which are stable throughout the synthesis. The remaining positions on both the sugar and the bases are protected in the same fashion as in DNA (1).

However, it should be noted that silyl groups require a specific deprotection step which makes the RNA synthesis more delicate to perform compared to the DNA synthesis. The presence of the protecting group at the 2' OH position creates a steric hindrance responsible for a lower coupling efficiency (95% to 98% for RNA versus 98% to 99% for DNA).

In consequence, PROLIGO recommands the use of purified RNA especially for RNA longer than 20 mers.

QUALITY CONTROL

PROLIGO routinely proposes custom synthesis of either crude or purified RNA ranging from 7 to 40 mers.

However, PROLIGO recommends, as explained above, purification for RNA superior to 20 mers.

When 1 to 5 OD is needed, purification is performed by PAGE whereas greater quantities require HPLC.

In addition, PROLIGO can synthesize short length RNAs, from 2 to 7 mers. This synthesis, as other modifications added to the ribo-oligonucleotide (fluorescein, phosphate, biotin, 2'0 Alkyl...), requires special synthesis and quality control procedures and consequently will be done upon specific quotation.

All RNA oligonucleotides are controlled on a denaturing polyacrylamide gel with a size marker.

Each RNA is delivered dried with a technical data sheet. The picture of the gel is available upon request.

Warning: RNA oligonucleotides must be handled with care since they are sensitive to RNase. RNA should be resuspended in sterile water and stored at - 20° C.

APPLICATIONS

RNA oligonucleotides are commonly used as a probe in Northern blot (the stability of the duplex RNA/RNA is superior to DNA/RNA > DNA/DNA) or in in situ hybridization.

More recently it has been shown that RNA oligonucleotides can be used in an antisense approach for regulating gene expression (2-4). Their specificity results from base pairing interaction in the heteroduplex between the DNA target and the RNA antisense sequence.

In addition, catalytic RNA or ribozyme (5,6) have within the nucleic acid sequence, a catalytic activity capable of cleaving the target.

Another application refers to crystallography studies of RNA oligonucleotides.

References

  1. S.L. Beaucage and R.P. Iyer (1992) - Tetrahedron 28, 2223.
  2. Weintraub H.M. (1990) - Antisense RNA and DNA. Sci. Am., 262, 40-46.
  3. Yu D, et al., (1996) - Bioorg Med Chem.; 4(10): 1685-1692.
  4. Helene C., Toulme J. (1990) - Nucleic Acids. Biochim. Biophys. Acta 1049, 99-125.
  5. Brown JW., (1998) - Nucleic Acids Res. 26(1): 353-354.
  6. Feig AL, et al., (1998) - Science; 279(5347): 81-84.

CONCLUSION

PROLIGO can supply the customer both crude and purified RNA and custom hybrid oligonucleotide DNA/RNA.

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