DNA aptamer-based rolling circle amplification product as a novel immunological adjuvant

Abstract

Several agonists to CD40 have shown to induce acquired immune responses. Here, we developed and evaluated the rolling circle amplification (RCA) products that are based on anti-CD40 DNA aptamers as a novel vaccine adjuvant. First, we developed DNA aptamers with specific binding affinity to chicken CD40 extra domain (chCD40ED). Next, we prepared the RCA products that consist of these aptamers to increase the spanning space and overall binding affinity to chCD40ED. Using 8 DNA aptamer candidates, 4 aptamer-based RCA products (aptamer RCAs) were generated, each consisting of two distinct aptamers. We demonstrated that all 4 aptamer RCAs significantly induced the signal transduction in chicken HD11 macrophage cell line (p < 0.05). Finally, we conjugated one of the aptamer RCAs (Aptamer RCA II) to M2e epitope peptide of influenza virus as a model hapten, and the immune complex was injected to chickens. Aptamer RCA II stimulated anti-M2e IgG antibody production to the level significantly higher as compared to the control (M2e epitope alone; p < 0.05). The results of our work suggest that aptamer RCA is a novel platform to boost the efficacy of vaccines, which might find broad applications to other antigens beyond M2e epitope evaluated in this study using chicken infection model.

Single-stranded DNA aptamer-based rolling circle amplification as anti-chicken Salmonella bacteriostatic

“…The isothermal RCA technique and protocol were used as described by Al-Ogaili et al . [ 10 ]. Briefly, 5' phosphorylated RCA template (equivalent to 2 pmol) was mixed with the primer (equivalent to 1 pmol) in 1×TE buffer (Promega, Cat# V6231, WI, USA).…”

“…Isothermal ɸ29 DNA polymerase with master mix (NEB, Cat# M0269L MA, USA) was used as the polymerization enzyme. The polymerization incubation condition was 30°C for 16 h [ 10 ]. After polymerization, the complementary spacer was added and annealed thermally (at 95°C for 5 min and 56°C for 1 min).…”

“…Recent developments in aptamer selection methodologies have shed new insights into the beneficial use of aptamers. Today, aptamers are beginning to replace monoclonal antibodies in several branches of biology and medicine [ 8 - 10 ].…”

A sensing system constructed by combining a structure-switchable molecular beacon with nicking-enhanced rolling circle amplification for highly sensitive miRNA detection

“…[45][46][47] Due to the high base-pairing fidelity of the polymerization reaction and high ratio of target-to-repeat units (hundreds to thousands), an RCA-based assay displays excellent specificity and prominent sensitivity. By specifically designing the circular template, repeat units can be customized as functional sequences, such as DNA aptamers, 48,49 DNAzymes 50,51 and restriction enzyme sites. 52,53 In this study, a powerful assay system for the sensitive detection of cancerrelated miRNA-21 is demonstrated based on a combination of a structure-switchable molecular beacon (SMB) with nickingenhanced rolling circle amplification (NRCA), into which the strand displacement process and RCA process are integrated.…”