Distance dependent photoacoustics revealed through DNA nanostructures

Abstract

Molecular rulers that rely on the Förster resonance energy transfer (FRET) mechanism are widely used to investigate dynamic molecular processes that occur on the nanometer scale. However, the capabilities of these fluorescence molecular rulers are fundamentally limited to shallow imaging depths by light scattering in biological samples. Photoacoustic tomography (PAT) has recently emerged as a high resolution modality for in vivo imaging, coupling optical excitation with ultrasound detection. In this paper, we report the capability of PAT to probe distance-dependent FRET at centimeter depths. Using DNA nanotechnology we created several nanostructures with precisely positioned fluorophore-quencher pairs over a range of nanoscale separation distances. PAT of the DNA nanostructures showed distance-dependent photoacoustic signal enhancement and demonstrated the ability of PAT to reveal the FRET process deep within tissue mimicking phantoms. Further, we experimentally validated these DNA nanostructures as a novel and biocompatible strategy to augment the intrinsic photoacoustic signal generation capabilities of small molecule fluorescent dyes.

DNA-based nanocarriers to enhance the optoacoustic contrast of tumorsin vivo

“…[34] Namely, NC6 and NC8 were assembled by heating to 85° for 5 min, cooling down from 85 to 65°C in 20 steps (1°C per step, 5 min each step) and cooling down from 65 to 25°C in 80 steps (0.5°C per step, 12 min each step). NC2 was assembled under the following protocol: [31,32] Optical absorbance and fluorescence measurements: Absorbance and fluorescence properties of the NCs were measured at 34°C. To compare the potential quenching effect of the NCQ+ versus NCQ-, the measurements were performed at matching IRDye 800CW concentration in PBS.…”

“…The OT response of the NCs at depths relevant for further in vivo studies was initially evaluated using tissue mimicking phantoms. Samples were encapsulated into agar-based tissue mimicking phantoms with defined optical properties (scattering coefficient of 5cm -1 and absorption coefficient of 0.05cm -1 at 700 nm) as previously described [31,32] (see Experimental Section).…”

“…[22][23][24][25][26][27] In the context of in vivo cancer imaging, DNA-NCs have been loaded with a variety of contrast agents for different modalities, including dyes for fluorescence imaging, [28] radioisotopes for SPECT [29] and PET, [30] as well as gold nanoparticles for OT. [23] Based on the knowledge that distance-dependent quenching of small molecule NIR dyes yields significant enhancement of OT signals, [31,32] we hypothesized that DNA-NCs could be used to construct biodegradable OT contrast agents with high optoacoustic signal generation and tumor uptake properties. Using precise positioning of IRDye 800CW [33] fluorophore molecules at very close proximities within DNA nanostructures, we obtained a substantial OA signal enhancement and tailored the effective size of these nanostructures to offer prolonged blood clearance times compared to the free dyes, thereby enhancing tumor uptake.…”

A rapid and concise setup for the fast screening of FRET pairs using bioorthogonalized fluorescent dyes

“…Fluorescently labeled complementary oligonucleotide strands provide an easy tool for investigating the suitability of novel dyes in FRET application. 33,34 When fluorescence modifications were situated in close proximity (5′ and 3′ matched) the estimated distance of the dyes was 18 Å; while positioned at distal ends (5′ and 5′ matched sequences) this distance was 51 Å ± 5 Å ( Fig. 3 and Table S1 †).…”

An active DNA-based nanoprobe for photoacoustic pH imaging

“…Their relative placement renders a change in the absorbance spectrum compared to the free F and Q, and consequently a modification in the wavelength-dependent PA response. 21 …”