Date/Time
Date(s) - 31/08/2021
10:30 am - 11:30 am

Title:                     Using Aptamers to Regulate Rolling CircleAmplification 

Date:                    Tuesday, August31, 2021

Time:                    10:30a.m.   

Zoom:                    link available form macchem@mcmaster.ca                               

Host:                    Dr.John Brennan 

Abstract:                    

Nucleic acids (NAs) are commonly used asmolecular recognition elements (MREs) for the detection of various analytes. Aptamers are one such NA species that are capable of adapting uniquesecondary and tertiary structures upon binding to non-NA targets.  A keyadvantage of using NAs is the ability t integrate isothermal NA amplificationstrategies.  For the development of biosensors and diagnostic assays,isothermal NA amplification methods such as rolling circle amplification (RCA)are often paired with aptamers for the rapid and sensitive quantification of avariety of non-NA biomarkers.  

In this presentation, I will describe the development of threenovel assay strategies for the simple, yet effective use of aptamers toregulate RCA for the real-time fluorescent detection of proteins and smallmolecules.  Phi29 DNA polymerase (phi29 DP) is one of the most usedpolymerases for RCA.  As phi29 DP exhibits difficulty processing DNAstrands that are bound to non-NA materials such as proteins, the first methodregulates RCA as target-binding of the aptamer blocks its use as a primer toinitiate amplification.[1]  While the first method is a turn-off sensor,the second method exploits the inherent 3’-exonuclease activity of phi29 DP togenerate a simple turn-on assay instead.[2]  Since target-bound aptamerswere shown to be resistant to exonuclease activity, phi29 DP preferentiallydigests target-free aptamers instead of target-bound aptamers.  Thetarget-bound aptamer could be liberated by a circular template and used as aprimer for RCA.  The final method used RecJf instead, which has5’-exonuclease activity, to modulate primer availability.[3] Target-binding conferred protection onto the aptamer from exonucleolyticdigestion by RecJf, and amplification of the protected aptamer could besubsequently initiated.

[1] R. M. Bialy, M. M. Ali, Y. Li, J. D. Brennan, Chem. Eur. J.2020, 26, 5085–5092.

[2] R. M. Bialy, Y. Li, J. D. Brennan, Chem. Eur. J. 2021,(Accepted) DOI: 10.1002/chem.202102975.

[3] R. M. Bialy, Y. Li, J. D. Brennan, Chem. Eur. J. 2021,(Submitted).