In Vitro Selection and Characterization of Cellulose-Binding RNA Aptamers Using isothermal Amplification

Abstract

We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust binding of cellulose in both the powdered and paper form, but did not show any significant binding of closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using glucosamine 6-phosphate to activate glmS ribozyme function.

Keywords:

• 3SR
• affinity chromatography
• glms ribozyme
• NASBA
• SELEX

Acknowledgments

We thank members of the Breaker Lab for helpful discussion. This work was supported by NIH grants DK070270 and HV28186. Research in the Breaker laboratory is also supported by the Howard Hughes Medical Institute.