An improved SELEX technique for selection of DNA aptamers binding to M-type 11 of Streptococcus pyogenes.

Streptococcus pyogenes is a clinically important pathogen consisting of various serotypes determined by different M proteins expressed on the cell surface. The M type is therefore a useful marker to monitor the spread of invasive S. pyogenes in a population. Serotyping and nucleic acid amplification/sequencing methods for the identification of M types are laborious, inconsistent, and usually confined to reference laboratories. The primary objective of this work is to develop a technique that enables generation of aptamers binding to specific M-types of S. pyogenes. We describe here an in vitro technique that directly used live bacterial cells and the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) strategy. Live S. pyogenes cells were incubated with DNA libraries consisting of 40-nucleotides randomized sequences. Those sequences that bound to the cells were separated, amplified using polymerase chain reaction (PCR), purified using gel electrophoresis, and served as the input DNA pool for the next round of SELEX selection. A specially designed forward primer containing extended polyA20/5Sp9 facilitated gel electrophoresis purification of ssDNA after PCR amplification. A counter-selection step using non-target cells was introduced to improve selectivity. DNA libraries of different starting sequence diversity (10(16) and 10(14)) were compared. Aptamer pools from each round of selection were tested for their binding to the target and non-target cells using flow cytometry. Selected aptamer pools were then cloned and sequenced. Individual aptamer sequences were screened on the basis of their binding to the 10 M-types that were used as targets. Aptamer pools obtained from SELEX rounds 5-8 showed high affinity to the target S. pyogenes cells. Tests against non-target Streptococcus bovis, Streptococcus pneumoniae, and Enterococcus species demonstrated selectivity of these aptamers for binding to S. pyogenes. Several aptamer sequences were found to bind preferentially to the M11 M-type of S. pyogenes. Estimated binding dissociation constants (Kd) were in the low nanomolar range for the M11 specific sequences; for example, sequence E-CA20 had a Kd of 7±1 nM. These affinities are comparable to those of a monoclonal antibody. The improved bacterial cell-SELEX technique is successful in generating aptamers selective for S. pyogenes and some of its M-types. These aptamers are potentially useful for detecting S. pyogenes, achieving binding profiles of the various M-types, and developing new M-typing technologies for non-specialized laboratories or point-of-care testing.

The Effects of SELEX Conditions on the Resultant Aptamer Pools in the Selection of Aptamers Binding to Bacterial Cells.

Aptamers of high affinity and specificity have a wide range of analytic and clinical applications. Selection of DNA or RNA aptamer molecules usually involves systematic evolution of ligands via exponential enrichment (SELEX), in which a random DNA or RNA library is incubated with a target molecule, and the oligonucleotides that bind the target are then separated from the nonbinders, PCR amplified, and used as refined libraries in the next round of selection. Conventional SELEX methodologies require the use of purified target molecules and their immobilization onto a solid support. However, purified targets from cells are not always available, and fixing the target to a support may alter its conformation. To overcome these problems, we have developed a SELEX technique using live bacterial cells in suspension as targets, for selecting DNA aptamers specific to cell-surface molecules. Through the selection of aptamers binding to Lactobacillus acidophilus and Streptococcus pyogenes, we report here optimization of this technique and show how varying selection conditions impact the characteristics of resultant aptamer pools, including the binding affinity, selectivity, and the secondary structures. We found that the use of larger starting library sequence diversity, gel purification of the subsequent pools, and the introduction of counter-selection resulted in a more efficient SELEX process and more selective aptamers. A SELEX protocol with lower starting sequence diversity, the use of heat denaturation, and the absence of counter-selection still resulted in high-affinity aptamer sequences specific to the target cell types; however, the SELEX process was inefficient, requiring 20 rounds, and the aptamers were not specific to the strain of the bacterial cells. Strikingly, two different SELEX methodologies yielded the same sequence that bound strongly to the target S. pyogenes cells, suggesting the robustness of the bacterial cell-SELEX technique.

DNA aptamers binding to multiple prevalent M-types of Streptococcus pyogenes.

This paper describes the selection of high affinity DNA aptamers binding to multiple M-types of the pathogenic species Streptococcus pyogenes (Group A Streptococcus or GAS). Unlike common aptamer selection techniques that use purified molecules of a monoclonal cell population as targets, this work has achieved the selection of aptamers against the various M-types of S. pyogenes. Cell mixtures containing equal numbers of the 10 most prevalent S. pyogenes M-types were incubated with 80-nucleotide DNA libraries, centrifuged, and washed to separate cell-bound from unbound DNA sequences. The DNA bound to the cells was amplified using the polymerase chain reaction, and the amplicons were tested for their binding to the target cells. The amplicons were also used as new DNA libraries for subsequent rounds of selection. Cloning, sequencing, and subsequent analysis of selected aptamers showed that they bind preferentially to GAS over other common and related bacteria. Resultant DNA aptamers showed strong and preferential binding to GAS, including the 10 most prevalent GAS M-types and another 10 minor M-types tested. Estimated K(d) values were in the range of 4 to 86 nM. Two aptamers, 20A24P and 15A3P (with estimated binding dissociation constants of 9 and 10 nM, respectively), are particularly promising. These aptamers could potentially be used to improve the detection of GAS, a pathogen that is the causative agent of many infectious diseases, most notably strep throat.

Selection and analytical applications of aptamers binding microbial pathogens.

DNA aptamers specifically recognizing microbial cells and viruses have a range of analytical and therapeutic applications. This article describes recent advances in the development of aptamers targeting specific pathogens (e.g., live bacteria, whole viral particles, and virally-infected mammalian cells). Specific aptamers against pathogens have been used as affinity reagents to develop sandwich assays, to label and to image cells, to bind with cells for flow-cytometry analysis, and to act as probes for development of whole-cell biosensors. Future applications of aptamers to pathogens will benefit from recent advances in improved selection and new aptamers containing modified nucleotides, particularly slow off-rate modified aptamers (SOMAmers).

Selection of aptamers against live bacterial cells.

Single-stranded DNA or RNA aptamer molecules have usually been selected against purified target molecules. To eliminate the need of purifying target molecules on the cell surface, we have developed a selection technique using live bacterial cells in suspension as targets, to select for ssDNA aptamers specific to cell surface molecules. Lactobacillus acidophilus cells were chosen to demonstrate proof of principle based on their high abundance of surface molecules (potential targets). Aptamer pools obtained after 6-8 rounds of selection demonstrated high affinity for and selective binding with L. acidophilus cells when tested via flow cytometry, microscopy, and fluorescence measurements. Out of 27 aptamers that were cloned and sequenced, one sequence, hemag1P, was found to bind to L. acidophilus much more strongly and specifically than other cells tested. This aptamer was predicted to have a tight hairpin secondary structure. On average, an estimated 164 +/- 47 aptamer molecules were bound to a target cell with an apparent K d of 13 +/- 3 nM. A likely putative molecular target of hemag1P is the S-layer protein on the cell surface.

Assays for cytokines using aptamers.

Aptamers are short nucleic acid sequences that are used as ligands to bind their targets with high affinity. They are generated via the combinatorial chemistry procedure systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have shown much promise towards detection of a variety of protein targets, including cytokines. Specifically, for the determination of cytokines and growth factors, several assays making use of aptamers have been developed, including aptamer-based enzyme-linked immunosorbent assays, antibody-linked oligonucleotide assay, fluorescence (anisotropy and resonance energy transfer) assays, and proximity ligation assays. In this article, the concept of aptamer selection using SELEX and the assay formats using aptamers for the detection of cytokines are discussed.