Non-SELEX: selection of aptamers without intermediate amplification of candidate oligonucleotides - Aptamers

Non-SELEX: selection of aptamers without intermediate amplification of candidate oligonucleotides
2006
Maxim V Berezovski, Michael U Musheev, Andrei P Drabovich, Julia V Jitkova and Sergey N Krylov
Nature Protocols
Aptamers are typically selected from libraries of random DNA (or RNA) sequences through systematic evolution of ligands by exponential enrichment (SELEX), which involves several rounds of alternating steps of partitioning of candidate oligonucleotides and their PCR amplification. Here we describe a protocol for non-SELEX selection of aptamers ? a process that involves repetitive steps of partitioning with no amplification between them. Non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), which is a highly efficient affinity method, is used for partitioning. NECEEM also facilitates monitoring of bulk affinity of enriched libraries at every step of partitioning and screening of individual clones for their affinity to the target. NECEEM allows all clones to be screened prior to sequencing, so that only clones with suitable binding parameters are sequenced. The entire protocol can be completed in 1 wk, whereas conventional SELEX protocols take several weeks even in a specialized industrial facility.
Introduction
DNA or RNA aptamers are single-stranded oligonucleotides that can bind proteins, small-molecule compounds and living cells with high affinity and specificity1, 2. Aptamers are promising affinity ligands with the potential to change the field of affinity probes and replace antibodies as diagnostic, analytical and therapeutic reagents3, 4, 5. Aptamers have indisputable advantages over antibodies owing to the ease and low cost of production, and the simplicity of chemical modifications and integration into different analytical schemes. The unique properties of aptamers have led to their application in many areas of bioanalytical and biomedical sciences. They have been successfully used in proteomics and development of bioanalytical assays6, inhibition of enzymes and receptors7, 8, development of artificial enzymes (ribozymes and aptazymes)9, target validation and screening for drug candidates10, 11, 12, cytometry and imaging of cellular organelles13, and development of biosensors14. Aptamers are gaining a reputation as therapeutic reagents for the treatment of different pathologies15. Their potential medical applications also include gene therapy and drug delivery to therapeutic targets16. Despite great promise and significant effort in the development of aptamers over a period of 15 yr, they have only been obtained for 100 protein targets17. This slow progress is largely due to the limitations of conventional technologies used for aptamer development.
Aptamers are typically selected from large libraries of random DNA or RNA sequences in a general approach termed systematic evolution of ligands by exponential enrichment (SELEX)18, 19. In essence, SELEX involves repetitive rounds of two alternating processes: (i) partitioning of aptamers from non-aptamers by separating target-bound DNA from free DNA; and (ii) amplification of aptamers by PCR (Fig. 1a).
Non-instrumental methods of partitioning, such as filtration and gel-electrophoresis, were initially used for SELEX, and still dominate the area20, 21, 22, 23, 24, 25. Because of high background (i.e., the high level of target-non-bound DNA collected along with target-bound DNA), SELEX based on conventional partitioning methods requires numerous rounds of selection (typically >10)26, 27, 28, 29, 30. As a result, SELEX based on conventional partitioning methods is a lengthy and resource-consuming process, which often leads to DNA structures that bind to the surfaces of the filters or chromatographic supports used in partitioning, rather than to the target28, 29, 30. Counter selection is successfully employed to eliminate such surface aptamers; however, it introduces additional rounds of selection, making the procedure even longer. Another disadvantage of too many rounds of selection is the limited number of unique aptamer sequences obtained at the output of conventional SELEX30. This disadvantage is critical for aptamer-based drug development, which requires as many 'lead molecules' as possible. Finally, if the efficiency of partitioning is too low, SELEX can fail to select aptamers.
Kinetic capillary electrophoresis (KCE) methods31, which started with the pioneering works of Heegaard and Whitesides on affinity capillary electrophoresis (ACE)32, 33, have established a new methodological platform for the partitioning of aptamers. So far, two distinct KCE methods have been used for the selection of aptamers: non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM)34, 35, 36, 37, 38 and equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM)37, 39. Bowser and co-authors were the first to use NECEEM in SELEX; they called the approach CE-SELEX34, 35. The partitioning efficiency of KCE methods exceeds that of conventional partitioning methods, such as filtration and column chromatography, by at least two orders of magnitude36. As a result, KCE methods decrease the number of rounds of SELEX from 10 (required by conventional partitioning techniques) to 1?3. In addition, KCE methods can be equally used for selection of aptamers and for measurements of their binding parameters: the equilibrium dissociation constant (K d), rate constants of complex formation (k on) and dissociation (k off), and the change of enthalpy (H) and entropy (S)40, 41. KCE methods have been demonstrated to facilitate the selection of 'smart' aptamers ? ligands with pre-defined binding parameters37, 39.
The outstanding partitioning capabilities of KCE methods have recently motivated us to attempt the selection of aptamers in a procedure that does not include intermediate amplification steps; we call this approach non-SELEX38 (Fig. 1b). Excluding repetitive steps of PCR accelerates the procedure of aptamer selection without compromising its efficiency. Omitting repetitive steps of PCR also excludes quantitative errors associated with the exponential nature of PCR amplification, thereby making non-SELEX a useful tool for studies of the properties of DNA libraries with respect to their interaction with targets. For example, non-SELEX can be used to accurately back-calculate the number of aptamer molecules in a naive DNA library. Furthermore, excluding repetitive steps of PCR allows us to avoid the bias related to differences in PCR efficiency with respect to different oligonucleotide sequences. Finally, non-SELEX can potentially provide a viable alternative to SELEX in the commercial development of aptamers42. It should be noted that the implementation of non-SELEX with currently available commercial CE instrumentation has a limitation: only a fraction of the collected ligands can be sampled for the next step of non-SELEX. This limitation requires that the fraction of aptamers in the naive library be no lower than 5 10-10 for the parameters used in this protocol. Our experience shows that the abundance of aptamers in the naive library is typically >5 10-10, thereby making the limitation less important.
In a recent proof-of-principle work, we demonstrated the use of NECEEM-based non-SELEX for the selection of DNA aptamers for h-Ras protein38. Here we describe a step-by-step protocol for this method (Fig. 2). As the method relies on electrophoretic separation of DNA?target complexes from free DNA, it is applicable to targets with relatively high molecular weights (e.g., proteins and peptides). In the pre-selection steps (Steps 1?10), migration times of the target, naive DNA library and DNA?target complex (if detectable) are measured. These parameters are used to determine the time window for collection of aptamers (we call this the aptamer-collection window). In addition, if the DNA?target complex is detectable, the bulk affinity of the naive library to the target is determined; this is later used to study the progress of aptamer selection. In the selection steps (Steps 11?19), candidate oligonucleotides are partitioned from the rest of the library by multiple steps of NECEEM with no PCR amplification between them. Fractions collected after every step of NECEEM partitioning are kept for subsequent analysis. In the analysis steps (Steps 20?33), all of the collected DNA fractions are concurrently amplified by PCR using the optimized number of PCR cycles, the strands of double-stranded PCR products are separated to obtain single-stranded aptamer pools and, finally, the bulk affinities of aptamer pools to the target are measured. In the cloning and sequencing steps (Steps 34?44), individual sequences from the best pool are amplified by bacterial cloning and asymmetric PCR, and their binding parameters (rate constants and equilibrium constants) to the target are measured. Only aptamers with suitable binding parameters are then sequenced.
The non-SELEX concept can also be used to develop a similar protocol for selection of RNA aptamers. NECEEM-based partitioning in the protocol can be replaced with partitioning by other KCE methods via minor changes in the procedure. In addition to selection of aptamers, non-SELEX provides the opportunity for selection of affinity ligands from DNA-tagged libraries of small molecules and peptides43, 44, 45. The DNA tags allow unequivocal identification of the corresponding small molecules when the DNA tags are PCR amplified and sequenced after partitioning. Due to the small size of the molecules with respect to that of the covalently-attached DNA tag, such libraries are expected to have electrophoretic properties identical to those of DNA libraries. SELEX is not applicable to such libraries as small molecules and peptides cannot be amplified by PCR.
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