SELEX

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Under the acronym SELEX ( Engl. : S ystematic E volution of L igands by EX ponential Enrichment to German: S ystematische E volution of L iganden by ex ponentielle enrichment ) refers to the molecular biology , a combinatorial method for the directed evolution of oligonucleotide strands , for example single-stranded DNA or RNA . These can bind specifically to selected targets as ligands .

properties

The oligonucleotide sequences obtained with the help of SELEX are called aptamers . The process is sometimes referred to as in vitro selection or in vitro evolution .
According to Ellington and Szostak, single-stranded DNA-based (ssDNA) aptamers are more suitable than RNA-based aptamers because of their higher stability with the same specificity.
SELEX ® is a registered trademark of Gilead Sciences .

The procedure

The SELEX procedure

At the beginning of the process, the synthesis consists of a very large number of different oligonucleotides, the so-called molecule library , also known as the molecule pool . From this pool of up to 10 16 and more different molecules, the molecules with the desired properties are isolated ( separated ) through targeted multiplication . The various molecules compete with each other and the molecules that best meet the requirement or task are selected and further multiplied. The selected and multiplied molecules are then subjected to another selection under varied and mostly more stringent conditions. Then only the best molecules are again specifically reproduced.
Through the repeated cycle of

  • Selection of binding RNA molecules, ( selection )
  • Separation of non-binding sequences ( separation )
  • enzymatic amplification of the RNA ligands interacting with the target molecule ( amplification ) and
  • (optional) mutation ,

one usually obtains sequences that bind very specifically and with high affinity to the desired target . This evolutionary approach is particularly easy to carry out with molecules such as DNA or RNA, since they can be reproduced relatively easily with the aid of the polymerase chain reaction . The cycle of selection - separation - amplification (mutation) is repeated up to twelve times.

The DNA / RNA library

3 'and 5' ends of a nucleic acid sequence

The single-stranded DNA starting library (ssDNA) is mostly produced by chemical DNA synthesis using automated solid-phase synthesis . A DNA synthesizer is set so that not a nucleotide is coupled at a certain point, but a mixture of all four nucleobases . The randomized sequence is produced in the middle between known sequences that are necessary for primer recognition.

The double-stranded DNA pool generated by PCR and transcribed into RNA can now be incubated in the next step of the selection process with the desired target molecule under carefully selected conditions .

The starting library usually contains a range of 20 to 220 randomized nucleotides. There are constant primer-binding 3 'and 5' sequences at the ends . In the constant range there is usually a promoter sequence that is essential for the in vitro transcription of the RNA.

The actual random sequence of n nucleotides is located between the two constant ends , which results in 4 n different possible sequences. In purely mathematical terms, a random sequence with n nucleotide elements results in:

  • n = 25 a library of approx. 10 15 sequences,
  • n = 50 a library of approx. 10 30 sequences,
  • n = 75 a library of approx. 10 45 sequences,
  • n = 100 a library of approx. 10 60 sequences possible.

One mole, i.e. 6.022 · 10 23 molecules of a 23mer - a nucleotide sequence with 23 randomized nucleobases - weighs approx. 7.5 kg. In order to statistically have one molecule of every possible variant, one needs 75 µg oligonucleotide. Only 3.75 mg oligonucleotide is required for 50-fold statistical coverage.
In contrast, the situation is completely different with longer-chain sequences. For a simple statistical coverage, after deducting the primers, you would need 13 kg for the 40mer, 16 kg for the 50mer and 32 kg for the 100mer . This is practically no longer possible from the 40mer .

The disadvantage of long random sequences is that the longer the random sequence, the smaller the section of possible sequences that a sample represents on a mg scale. The advantage of long random sequences , on the other hand, is that a single 100-mer already contains 75 different 25-mer sequences or 50 different 50- mer sequences. This can significantly accelerate the selection process.

selection

The incubation between the nucleotide sequences and the target structures (targets) can take place in the immobilized state or in solution. The forces of interaction between the multitude of nucleotide sequences and the target structures are very different. The binding of the nucleotides, based on the lock and key principle , is extremely dependent on their secondary structure . The secondary structure in turn depends very strongly on the sequence, that is to say the order of the nucleobases adenine , guanine , cytosine , thymine (in the case of DNA) or uracil (in the case of RNA).

Helices , triple helices , hairpin loops , pseudo-knots and G-quartets are described as secondary structures .

Separation

The sequences that are only weakly bound or not bound at all on the targets are separated (separated) from the bound sequences by means of different washing steps. The separation is usually carried out with the aid of affinity chromatography . The bound sequences are eluted and amplified with the PCR.

Amplification

The amplification is carried out by reverse transcription (only for RNA) and the polymerase chain reaction (PCR). This creates a pool of DNA or RNA molecules with - compared to the original molecule pool - improved binding properties to the ligand. This step closes the cycle and is now usually repeated eight to twelve times until a desired aptamer is obtained or only a few different sequences are enriched. By cloning and sequencing of the systematic evolution resulting monoclonal aptamers upon completion. A wide variety of molecules such as simple organic compounds, proteins or whole cells can be considered as possible target molecules.

mutation

As already described for the molecule library, a complete randomization of a 100-mer oligonucleotide with 10 60 (= 4 100 ) variants cannot be realized. You can only produce about 10 16 molecules. The most active oligonucleotides isolated from this limited pool can easily be modified by mutagenic PCR for further improvement. In this case, one also speaks of post-randomization . The PCR conditions are set so that the polymerase makes mistakes during the copying process. An error rate of, for example, 1 per 20 base pairs can be set by choosing the conditions.

Post-randomization is only recommended after several runs of the selection-separation-amplification cycle . Strictly speaking, it is not a separate process step, but a modification of the amplification step.

history

The SELEX method was developed at the same time in 1990 by Larry Gold and Andrew E. Ellington. Both approaches are based on the principle of selective recognition of target structures, mediated by the 3-dimensional shape of single-stranded nucleic acids. Gold used here single-stranded DNA ( single beach DNA , ssDNA), Ellington contrast, RNA with a defined surface structure.

Importance of the procedure

The selection of new enzymes based on RNA and DNA, the so-called ribozymes , has moved into the focus of research. The catalytically active RNA that folds into specific three-dimensional shapes, as one of proteins based enzyme reactions catalyze . In addition, like an antibody , the ribozymes can molecularly bind small molecules or proteins in a highly specific manner.
The production of these highly specific receptors, known as aptamers, on a DNA or RNA basis, which can bind to medically relevant targets , is the goal of SELEX.

The process is used to develop aptamers with extremely high binding affinity to different targets. Small molecules such as ATP , adenosine and proteins such as prions and signal molecules such as Vascular Endothelial Growth Factor (VEGF) are also targets for aptamers.

In the field of oncology , aptamers are very promising ligands.

The aptamer pegaptanib , which binds to VEGF, is approved for the treatment of age-related macular degeneration (AMD).

It should be noted, however, that extremely high binding affinities in the sub-nanomolar range do not necessarily increase the specificity for the target molecule. The so-called off-target binding has significant clinical effects.

The development and production costs for biosensors based on aptamers are estimated in the literature to be considerably lower than those based on proteins (e.g. antibodies).

Molecular Modifications

The use of 2'-fluoro- or 2'-amino-mononucleotide triphosphate-modified RNA libraries enables ribonuclease- resistant aptamers to be obtained. Aptamers modified in this way have the potential to function as probes in biological fluids ( blood , urine ).

Individual evidence

  1. ↑ a b A. D. Ellington et al .: In vitro selection of RNA molecules that bind specific ligands. in Nature , 346/1990, pp. 818-822
  2. ↑ a b C. Tuerk et al: Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. In: Science 249/1990, pp. 505-510.
  3. ^ AD Ellington, JW Szostak: Selection in vitro of single-stranded DNA molecules that fold into specific ligand-binding structures. In: Nature 355/1992, pp. 850-852
  4. ↑ a b c d e State Environment Agency NRW: 2006: Antibiotics, resistances and bacteria in sewage treatment plants.  ( Page no longer available , search in web archives )  Info: The link was automatically marked as defective. Please check the link according to the instructions and then remove this notice. (PDF; 4.2 MB) 2006@1@ 2Template: Toter Link / www.lanuv.nrw.de  
  5. ↑ a b Sabine Kainz: Selection and characterization of aptamers, specific for the virus of infectious bursitis. Dissertation, Hamburg 2005
  6. ↑ a b c University of Marburg: Use of the PCR in the in vitro random selection ( Memento of the original from September 3, 2016 in the Internet Archive ) Info: The archive link was inserted automatically and has not yet been checked. Please check the original and archive link according to the instructions and then remove this notice. @1@ 2Template: Webachiv / IABot / online-media.uni-marburg.de
  7. ↑ DP Bartel, JW Szostak: Isolation of new ribozymes from a large pool of random sequences. In: Science 261/1993, pp. 1411-1418
  8. ↑ AA Beaudry, GF Joyce: Directed evolution of an RNA enzyme. In: Science 257/1992, pp. 635-641
  9. ^ N. Leontis et al: The non-Watson-Crick base pairs and their associated isostericity matrices. In: Nucleic Acids Research , 30/2002, pp. 3497-3531.
  10. ^ BE Eaton et al.: Ribonucleosides and RNA. In: Annual Review of Biochemistry . 64/1995, pp. 837-863.
  11. ↑ F. Jiang et al .: Structural basis of RNA folding and recognition in an AMP-RNA aptamer complex. In: Nature 382/1996, pp. 183-186.
  12. ^ Woese CR, Architecture of ribosomal RNA: constraints on the sequence of "tetra-loops. In: Proceedings of the National Academy of Sciences . 87/1990, pp. 8467-8471.
  13. ↑ F. Jarosch et al .: In vitro selection using a dual RNA library that allows primerless selection. In: Nucleic Acids Research. 34/2006, p. 86.
  14. ↑ a b Michael Blank: Systematic evolution of DNA aptamers for the characterization and diagnosis of pathologically changed endothelial cells in tumors and inflammatory regions of the rat brain . Tübingen 2002, DNB  965284956 , urn : nbn: de: bsz: 21-opus-5841 (dissertation, University of Tübingen).
  15. ↑ T. Dieckmann et al.: Solution structure of an ATP-binding RNA aptamer reveals a novel fold. In: RNA 2/1996, pp. 628-640.
  16. ^ DE Huizenga, JW Szostak: A DNA aptamer that binds adenosine and ATP. In: Biochemistry . 34/1995, pp. 656-665.
  17. ↑ DH Burke, L. Gold: RNA aptamers to the adenosine moiety of S-adenosyl methionine: structural inferences from variations on a theme and the reproducibility of SELEX. In: Nucleic Acids Research. 25/1997, pp. 2020-2024.
  18. ↑ R. Mercey et al .: Fast, reversible interaction of prion protein with RNA aptamers containing specific sequence patterns. In: Archives of Virology. 151/2006, pp. 2197-2214.
  19. ↑ a b H. Ulrich et al .: DNA and RNA aptamers: from tools for basic research towards therapeutic applications. In: Combinatorial Chemistry & High Throughput Screening . 9/2006, pp. 619-632.
  20. ↑ CS Ferreira et al.: DNA aptamers that bind to MUC1 tumor marker: design and characterization of MUC1-binding single-stranded DNA aptamers. In: Tumor Biology . 27/2006, pp. 289-301.
  21. ↑ D. Vavvas, DJ D'Amico: Pegaptanib (Macugen): treating neovascular age-related macular degeneration and current role in clinical practice. In: Ophthalmology Clinics of North America. , 19/2006, pp. 353-360.
  22. ↑ JM Carothers et al .: Aptamers selected for higher affinity binding are not more specific for the target ligand. In: Journal of the American Chemical Society. 128/2006, pp. 7929-7937.
  23. ↑ B. Eaton et al. In Annual Review of Biochemistry . 64/1994, pp. 837-863
  24. ↑ W. Pieken in Science , 253/1991, pp. 314-317

literature

Web links