In the name of God Aptamer in-vitro selection SELEX & Non-SELEX

1. In the name of GodAptamer in-vitro selection SELEX & Non-SELEX Maryam Tabarzad ShahidBeheshti University of Medical Science 1392

2. History • Aptamers are synthetic, highly structured, single stranded DNA/RNA ligands • Term “Aptamer”(Ellington&Szostak 1990) : aptus(to fit) + mer(oligo) • The first SELEX experiment on single- stranded oligonucleotides was published by Tuerk and Gold in 1990 • 'Systematic evolution of ligands by exponential enrichment' (SELEX) • A protocol in which vast libraries of single-stranded oligonucleotides are screened for desired activities

3. Systematic Evolution of Ligands by Exponential enrichment

5. SELEX components • Oligonucleotide random pool • DNA/RNA • Modified nucleic acids

6. DNA or RNA • RNA : in vitro transcription • ssDNA: • Asymmetric PCR • Biotin-streptavidin separation • Lambda exonuclease digestion • Size separation on denaturing-urea PAGE

7. SELEX components (cont.) • Target • Small molecules • Bimolecules • Whole cells • Whole organisms

8. SELEX components (cont.) • Separation techniques • Filtration (nitrocellulose, Dialysis) • Chromatography (size exclusion, Affinity) • Electrophoresis (PAGE, Agarose, CE) • Magnetic beads • Precipitation (Immunoprecipitation, centrifuge) • SPR • AFM

11. Targets of selection

12. Negative SELEX To exclude adsorbed ssDNA or RNA by the matrixes used for immobilizing targets with the purpose of gaining aptamers that can only adsorb the targets

13. Counter SELEX Alike to the negative SELEX In order to get aptamers with stronger specificity Excluding ssDNA or RNA molecules with coaffinity to the similar molecules of the targets. Established by Jenison and his fellows(theophylline , caffeine )

14. Subtractive SELEX The same purpose as counter SELEX To improve the selectivity of aptamers The main difference between them lies in the targets of the process. The targets of complex targets Eliminates the ssDNA or RNA sequences that can bind the no purpose part of the complex target. by Chinese researcher Wang (capability to distinguish differentiated PC12 cells from normal PC12 cells)

15. Toggle SELEX A method to deal with several kinds of targets at the same time The different rounds of the screening cycles focus on different targets according to the strategy during this toggle SELEX.

16. TECS- SELEX Target Expressed on Cell Surface-SELEX

17. Blended SELEX Other molecules, which can lead the oligonucleoride chain to the specific region of the target, are mixed to the screening pool. This method was established by Smith first, For drug discovery They connected valylphosphonate moiety (valP, an inhibitor of human neutrophil elastase) with the 5’ linker of a DNA splint oligonucleotide, which can combine with the end of the RNA pool according to the principle of complementary base pairing. This inhibitor could be joined to the random sequence to form a “blended pool”

18. Conditional SELEX Method for producing nucleic acid ligands that generate a signal, or cause a decrease in the level of a signal, in the presence of a target molecule or an environmental stimulus. To measure the concentration of a target molecule or detect and quantitate an environmental stimulus.

19. Regulated aptamers

20. Mirror-image SELEX The improvement of their stability and half-lives of DNA and RNA in vivo becomes the key point Stable aptamers can be obtained by Spiegelmer Technology. Screens aptamers of chiral target from dextrorotatory oligonucleotide pool Then, the corresponding levorotatory oligonucleotide, called Spiegelmers, is synthesized

21. Spiegelemer Spiegelmers have a good stability in vivo for not being able to be degraded by ribonuclease. This method only suits for peptides and small molecules with optical activity. When targets do not have optical activity, this method could not be used anymore. Spiegelmer cannot be amplified by PCR, it loses the advantage to be quickly obtained plentiful of aptamer in vitro.

22. Random oligonucleotide Pool

23. Photo SELEX • A screening method that uses the photosensitive nuclear acids’ ability of covalent cross-link with other molecules under a certain wavelength’s light • The selectivity of the aptamers would be higher • The false positive of this method is also higher • 5-IU or 5-BrdU is mixed into the screening pool, then the pool incubates with the target, the mixture is irradiated by ultraviolet radiation to make the cross-link reaction happen • The wavelength of the UV radiation and the irradiation time should be optimized to ensure that only specific sequences could react with the targets. • This method was first used in 1995 by Jensen, who obtained aptamers for Rev of HIV-1 by using 5-IU

24. Genomic SELEX Uses the whole genome of a certain organism as the screening pool Bioactive molecules as the targets Great potential in the research of the interaction of bioactive molecules and nuclear acids Study the regulation networks between protein and nuclear acid in proteomics Include all the gene sequences and the intron sequences of the genome

25. cDNA-SELEX At 1995, another library modification strategy was proposed by Dobbelsteinet al. use of total cell RNA from Human B-cell lymphoma Revealed three sites on 28S ribosomal RNA that have the potential to interact with L22

26. In Vivo SELEX In 1997 the first report of a selection made inside mammalian cells was done by Coulter et al. uses transient transfection and an iterative procedure to enrich RNA-processing signals in culture To find splicing enhancers sites

27. Expression Cassettes SELEX Peviously isolated aptamer to the E2F1 transcription factor was coupled to a Pol III promoter as an expression unit (or cassette) in a plasmidic DNA. The construct contained a promoter, a tRNA sequence and the aptamer flanked by randomized regions.

28. Expression Cassettes SELEX When the transcript was generated, tRNA structure stabilized the aptamer and the randomized stretches formed a stem flexible enough to allow the formation of the proper configuration for target recognition. This expression cassette yielded RNAs that bind E2F with high affinity without sacrificing its structure and which can be stably expressed at high levels in mammalian cells

29. Multiple pools SELEX A method that combines aptamers from different pools via a certain way Screens the combinational production again in order to obtain multiple functional aptamers Presently, two modes have been established

30. Chimeric SELEX Burke fused pairs of aptamers previously selected and the results show that the binding ability of the new aptamers to both the targets were reduced. Applying dual selection pressure to recombined populations yielded the combinations that were best suitable for binding both targets. The method can generate dual-function aptamers for a wide variety of applications, including catalysis, novel therapeutics, and studies of long-range RNA structure

31. Multi Stage SELEX • To study of binding mechanism of aptamers and their targets • Two nucleic acid pools of N40 and N60 were founded to screen aptamers for cibacron blue and cholicacid • Stage one starts on the selection of parental aptamers from randomized libraries. • Stage two, a counter-selection step is used to avoid cross-reaction between each selection target (cibacron blue and cholic acid). • Stage three, obtained aptamers are fused to each other and reselected to isolate the most affinity allosteric pairs. • Stage four was to separate binding regions and returned to the counter-selection like stage two. • In stage five, new allosteric-DNA combinations are re-joined and re-selected to later be cloned and characterized

32. Tailored SELEX Classical screening of aptamers must truncate the primers after sequencing and this must affect the selectivity of the aptamers, especially the aptamers with short random areas. This technique decreases the number of the oligonucleotides of the primers according to the principle of complementary base pairing. At the time of amplification, primers can be added to both the ends by a bridge sequences; after the amplification, these primers can be eliminated by alkali and the new pool for the next cycle is still with short stable sequences.

33. Primer-Free SELEX Wen used a genomic library derived from the bacteriophage, and the gene 5 protein (g5p) from the phage as the target protein to obtain aptamers by means of primer free SELEX. Primer sequences are removed from the genomic pool before incubation of the target protein and are then regenerated to allow amplification. A key step in the regeneration of primer-annealing sequences is to use thermal cycles of hybridization-extension, with the sequences from unselected pools as templates. Pan et al reported their work about minimal primer and primer-free SELEX protocols for screening of aptamers from random DNA libraries as well.

34. Signaling Aptamers Basic design of a SELEX for aptamer beacons starts with a library that had been amplified using a 5′-labeled primer (fluorescein) Then hybridized with a capture oligonucleotide that is biotynilated on the 3′-end and coupled to a particular quencher in the 5′-end. Hybrids are recovered using streptavidin beads and mixed with the target In such way that only those sequences forming specific interactions are released and dequenched. Each aptamer has the property of emitting a signal proportional to target concentration.

35. Partitioning Methods

36. FluMAG SELEX Stoltenburget al. 2005 Used fluorescent labeling and an target immobilized over magnetic beads The possibility of applying them as biosensors useful in clinical approaches

37. Deconvolution SELEX Gold et al. 1998 Red Blood Cells Specific aptamers to cell surface markers

38. MonoLex affinity chromatography followed by physical fragmentation of the resin column Nitsche et al. Complete Vaccinia virus particles

39. CE SELEX • Capillary electrophoresis has been high efficiency and widely used in the analysis of nuclear acids and proteins. • Capillary electrophoresis was used in the screening of aptamers by Mendonsa and Bowser firstly at 2004 • The reasons for the high efficiency of CE SELEX are as follows: • first, random sequences with stronger capacity will be obtained in each cycle because of the high efficiency of capillary electrophoresis • second, the target in this method is in solution, without the interruption of the matrix, the requirement of negative SELEX will be eliminated

40. NanoSelection® (nM-AFM SELEX) Atomic force and fluorescence microscopy were combined with small copy number PCR by Penget al. in 2007 The possibility to isolate individual aptamers in a single selection cycle

41. Microfluidic chips A little sample of target-DNA mixture (up to 5 nL) was injected into a capillary and separated with a high voltage setting the bases of microfluidics SELEX Micro-magnetic device (MMS)

42. SPR Surface Plasmon Resonance Flow cytometeryCell SELEX

44. Non-SELEX High efficient and fast method for aptamers’ screening. The key point of this method is that it omits the magnification step by PCR. The random sequences with binding ability separated from the previous circle are used directly in the next one. The screening process claims higher efficiency of the separation step, while capillary electrophoresis is a good choice.

45. Nonequilibrium capillary electrophoresis of equilibrium mixtures is a new separation-based affinity method. NECEEM

46. Automated SELEX Cox set up a robotic work station configuration based on an augmented BeckmanuBiomek 2000 Pipetting robot for screening aptamers in 1998 and obtained aptamers for lysozyme The described robotic work station can carry out eight screenings in parallel and will complete approximately 12 rounds of selection in 2 days. Furthermore, the automatic work station was improved by containing a part of the generation of protein targets directly transcribed and translated from the respective gene in vitro on the robotic work station. This should further accelerate aptamer screening for proteins and increase the utility of aptamers as reagents in proteome analysis

47. Half automated SELEX As the screening conditions are not the same for all the aptamers, the automated system should be designed with some flexibilities such as the adjustment of the buffer components, the change of the time of incubation, and the optimization of the conditions for PCR. The half-automated screening platform developed by Eulberg in 2005 can meet the claims above to some degree. This system based on Amp 4200E (MWG Biotech Ebersberg, Germany) co-worked with ultrafiltration, fluorescence detection, and semiquantitative PCR. With this system, they obtained RNA aptamer for substance P

48. Bioinformatics Approaches for SELEX In silico analysis has also been used to study the behavior of nucleic acid populations during SELEX