Application of high throughput sequencing in selection of RNA aptamers

Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)




Philip N. Borer

Second Advisor

John M. Belote

Third Advisor

Mark S. Brainman


RNA aptamers, Oligonucleotide, Selection cycles, High throughput sequencing, FIXa

Subject Categories



Aptamers are discovered from random oligonucleotide libraries by an in vitro selection process, usually called SELEX (Systematic Evolution of Ligands by EXponential enrichment). The conventional SELEX method usually takes multiple rounds of selection and amplification, which are labor-intensive and time-consuming. Recently developed high-throughput sequencing (HTS) technologies could offer great advantages in aptamer discovery. Here we demonstrated the application of HTS in the selection of RNA aptamers, which dramatically reduced the number of selection cycles and greatly shortened the discovery process. Over-represented libraries were selected against the target, followed by HTS, where aptamer candidates were revealed by counting their frequency of occurrence. In fact, the known RNA aptamer for human factor IXa (FIXa) was successfully selected from the over-represented m=16 chimeric library after a single round of selection. This successful single-pass aptamer selection could be attributed to three factors: over-representation in the initial library; efficient separation in the partitioning step and the massive number of sequences generated by HTS. An unprecedented family of aptamers for FIXa with relatively high affinity ( K d = 220∼1100 nM) was indentified from the sparsely represented RNA library with m=35 in two rounds of selection, where the first round of selection was performed to reduce complexity and give the subpool over-representation. Three aptamers demonstrated substantial inhibition of FIXa activity. These new m=35 RNA aptamers are all consistent with an aptamer motif, Q1-L1-Q2-L2-Q3-L3-Q4-Tail, and they probably form a quadruplex. Although observed with low counts, the top sequences selected for FIXa from an m=22 RNA library could form the same qudraplex motif as the new m=35 RNA aptamer sequences. It appears that the qudraplex motif is favored by FIXa under current library design and selection conditions.


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