In Vitro antibacterial effects of antilipopolysaccharide DNA aptamer-C1qrs complexes

Abstract

DNA aptamers were developed against lipopolysaccharide (LPS) from E. coli O111:B4 and shown to bind both LPS and E. coli by a colorimetric enzyme-based microplate assay. The polyclonal aptamers were coupled to human C1qrs protein either directly using a bifunctional linker or indirectly using biotinylated aptamers and a streptavidin-C1qrs complex. Both systems significantly reduced colony counts when applied to E. coli O111:B4 and K12 strains across a series of 10x dilutions of the bacteria in the presence of human serum; it was diluted 1: 10(3) in order to avoid significant bacterial lysis by the competing alternate pathway of complement activation. A number of candidate DNA aptamer sequences were cloned and sequenced from the anti-LPS aptamer library for future screening of antibacterial or "antibiotic" potential and to aid in eventual development of an alternative therapy for antibiotic-resistant bacterial infections.

Aptamer–biotin–streptavidin–C1q complexes can trigger the classical complement pathway to kill cancer cells

“…Unlike the bacterial killing experiments of Bruno et al (2008c) in which precipitous drops in colony counts were noted as a function of increasing aptamer-C1qrs conjugate to bacteria ratios, lysis of cancer cells is less dramatic. The decreased efficacy of aptamer-mediated and antibodymediated cancer cell killing via serum complement proteins is probably due to a combination of MUC1 surface antigen shedding (Chan et al 1999;Moreno et al 2007;Storr et al 2008;Rubinstein et al 2009) during the experiment and membrane-bound complement regulatory molecules such as CD46, CD55, and CD59 on normal and cancer cells (Halulinen and Meri 1998;Farkas et al 2002;Donin et al 2003;Yan et al 2008).…”

“…It may seem superficially obvious that aptamers could take on many of the physiological or immune system functions of antibodies such as opsonization of encapsulated bacteria (Bruno et al 2008b) or complement-mediated target cell lysis (Bruno et al 2008c), but one should consider the complex conformational changes that occur in some of these processes. For example, C1q must bind to two adjacent IgG antibodies or one IgM on a target cell surface and then undergo conformational changes with C1r and C1s bound to it in order to induce the esterase activity which sets off the remainder of the complement cascade and culminates in cell lysis.…”

“…In microbiological applications, DNA aptamer-Fc conjugates are being used to emulate antibodies by enhancing phagocytosis (Bruno et al 2008b). In addition, lipopolysaccharide covalent DNA aptamerC1qrs conjugates and similar streptavidin (SAv)-biotinlinked complexes have recently been coupled to the classical complement cascade to kill Gram negative bacteria (Bruno et al 2008c), further demonstrating aptamer likeness to antibody functionality.…”

Aptamers against pathogenic microorganisms

“…A DNA aptamer against lipopolysaccharide of E. coli O111:B4 was selected (Bruno et al, 2008b) and applied as a basis for a conjugate with human C1qrs protein which activates the complement system responsible for non-specific resistance to bacteria. The resultant conjugate demonstrated an antibacterial activity in the presence of complement proteins, and could be considered as a prospective antibacterial drug.…”

A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane proteins

“…Bruno et al have recently evolved two DNA aptamers; one against E. coli O111 lipopolysaccharides [21] that were employed in a displacement voltammetric assay [22] and the other against outer membrane proteins (OMPs) [23]. In the latter work several aptamers were obtained and tested for competitive displacement FRET assays.…”