Efficient Selection of Biomineralizing DNA Aptamers Using Deep Sequencing and Population Clustering

Abstract

DNA-based information systems drive the combinatorial optimization processes of natural evolution, including the evolution of biominerals. Advances in high-throughput DNA sequencing expand the power of DNA as a potential information platform for combinatorial engineering, but many applications remain to be developed due in part to the challenge of handling large amounts of sequence data. Here we employ high-throughput sequencing and a recently developed clustering method (AutoSOME) to identify single-stranded DNA sequence families that bind specifically to ZnO semiconductor mineral surfaces. These sequences were enriched from a diverse DNA library after a single round of screening, whereas previous screening approaches typically require 5-15 rounds of enrichment for effective sequence identification. The consensus sequence of the largest cluster was poly d(T)30. This consensus sequence exhibited clear aptamer behavior and was shown to promote the synthesis of crystalline ZnO from aqueous solution at near-neutral pH. This activity is significant, as the crystalline form of this wide-bandgap semiconductor is not typically amenable to solution synthesis in this pH range. High-resolution TEM revealed that this DNA synthesis route yields ZnO nanoparticles with an amorphous-crystalline core-shell structure, suggesting that the mechanism of mineralization involves nanoscale coacervation around the DNA template. We thus demonstrate that our new method, termed Single round Enrichment of Ligands by deep Sequencing (SEL-Seq), can facilitate biomimetic synthesis of technological nanomaterials by accelerating combinatorial selection of biomolecular-mineral interactions. Moreover, by enabling direct characterization of sequence family demographics, we anticipate that SEL-Seq will enhance aptamer discovery in applications employing additional rounds of screening.

Trends in aptamer selection methods and applications

“…Aptamer candidates can be determined by the highthroughput sequencing at much earlier cycles of a selection starting from the naïve/non-enriched pool itself whereas the cloning based aptamer identification obliges evolution of the selection pool down to a few sequences. Consistent with the theory, Bawazer et al 71 implemented the sequence similarity (motifs or consensus sequences) and the high abundance rate of the sequences in the aptamer selection, in which aptamers specific to zinc oxide (ZnO) semiconductor mineral surfaces (bio-mineralizing DNA aptamers, facilitating ZnO mineralization from a hydrated Zn(NO3)2 precursor) For instance, Wilson et al 27 recently reported bivalent thrombin aptamers selected at single cycle using 454 GS FLX NGS platform that enabled comprehensive data analysis obtained from the sequential dissociation rounds.…”

Poly‐cytosine DNA as a High‐Affinity Ligand for Inorganic Nanomaterials

“…This may explain why the above aptamers are made of simple repeating sequences. [11,12] Therefore,i nstead of performing aptamer selections,D NA homopolymers might be ag ood starting point for surface-binding DNA. Herein, we communicate aquite surprising yet useful finding that poly-cytosine (poly-C) DNAbinds strongly to many common inorganic surfaces.…”

Selection and Screening of DNA Aptamers for Inorganic Nanomaterials

“…Gerdon and co‐workers used a precipitation SELEX method to isolate DNA aptamers for calcium phosphate . A single round of aptamer selection was performed by Morse and co‐workers on ZnO, and a simple T 30 DNA sequence was concluded to be its aptamer . By a screening method, however, poly‐C DNA was found to adsorb more strongly than poly‐T DNA .…”

“…With a high surface binding affinity, such DNA can also be useful for controlling the growth of nanoparticles. For example, attempts have been made with mineralization using ZnO . The much more systematically studied examples are the growth of gold nanostructures (Figure C) .…”

“…Beyond DNA immobilization, surface binding aptamersa re also potentially useful for other applications such as dispersing nanoparticles, [27] stabilization and sorting of nanomaterials, [28] DNA-directed mineralization, [25,29] and for understanding fundamental interactions between surfacesa nd DNA. However,s uch aptamersh ave not caught much attention in application so far.T he problem seems to be the lack of high quality aptamers from the typical aptamer selection process.…”