Sequence-selective purification of biological RNAs using DNA nanoswitches

Abstract

Nucleic acid purification is a critical aspect of biomedical research and a multibillion-dollar industry. Here we establish sequence-selective RNA capture, release, and isolation using conformationally responsive DNA nanoswitches. We validate purification of specific RNAs ranging in size from 22 to 401 nt with up to 75% recovery and 99.98% purity in a benchtop process with minimal expense and equipment. Our method compared favorably with bead-based extraction of an endogenous microRNA from cellular total RNA, and can be programmed for multiplexed purification of multiple individual RNA targets from one sample. Coupling our approach with downstream LC/MS, we analyzed RNA modifications in 5.8S ribosomal RNA, and found 2'-O-methylguanosine, 2'-O-methyluridine, and pseudouridine in a ratio of ~1:7:22. The simplicity, low cost, and low sample requirements of our method make it suitable for easy adoption, and the versatility of the approach provides opportunities to expand the strategy to other biomolecules.

Graphical abstract

Figure 1

DNA nanoswitch overview and workflow of purification (A) Challenge of single species RNA purification. (B) The DNA nanoswitch converts from a linear to looped form in the presence of target RNA. (C) The purification workflow consists of three major steps: RNA capture, RNA-nanoswitch isolation, and final RNA cleanup.

Figure 2

Validation of RNA purification (A) Overview of entire purification process. (B) Validation and quantification of the recovery for miR-16 in buffer, compared with no template control (NTC). (C) Validation and quantification of the recovery for miR-16 spiked into total RNA. Bar graphs show mean and standard deviation from three purification replicates.

Figure 3

Purification of endogenous miR-16 from total RNA (A) Illustration showing purification of miR-16 from cellular total RNA using nanoswitch method and commercially available beads-based method. (B) Recovery of miR-16 from MCF-7 total RNA and elimination of nonspecific rRNAs using DNA nanoswitches and beads-based method. Purification was performed in duplicate for miR-16 measurement and triplicate for nonspecific RNA, with bars representing mean and standard deviation. Non-amplifying wells were excluded from analysis. (C) Comparison of DNA nanoswitch method with commercial beads-based method.

Figure 4

Multiplexed purification: concept, validation, and application to rRNAs (A) Concept of multiplexed purification. (B) Validation of multiplexed purification using an mRNA fragment and a microRNA. (C and D) Application of multiplexed purification in selecting 5S and 5.8S rRNA from HeLa cell total RNA. (E) Validation of purified rRNAs by redetecting with nanoswitches.

Figure 5

Purification of RNA with chemical modification (A) The process of purification and verification by LC-MS/MS of modified RNAs. (B) Modification probing of the purified RNA molecules by LC-MS/MS. Left: m^4,4C modification standard, middle: synthetic RNA with modification, and right: purified RNA from 10 nM sample. The target RNA molecule has an m^4,4C modification on a cytosine (sequence shown above graph on right, ∗ indicates position of modification). (C) Purification of 5.8S rRNA from HeLa cell total RNA and illustration of known modifications and their positions. (D–F) Probing 2′-O-methyluridine (Um), 2′-O-methylguanosine (Gm), and pseudouridine (ψ) modifications in the purified 5.8S rRNA by LC-MS/MS and (G) mole ratio of the three modifications Um, Gm, and ψ. Bars represent mean and standard deviation from two technical replicates of a single purified sample.