Monitoring integrity and localization of modified single-stranded RNA oligonucleotides using ultrasensitive fluorescence methods

Abstract

Short single-stranded oligonucleotides represent a class of promising therapeutics with diverse application areas. Antisense oligonucleotides, for example, can interfere with various processes involved in mRNA processing through complementary base pairing. Also RNA interference can be regulated by antagomirs, single-stranded siRNA and single-stranded microRNA mimics. The increased susceptibility to nucleolytic degradation of unpaired RNAs can be counteracted by chemical modification of the sugar phosphate backbone. In order to understand the dynamics of such single-stranded RNAs, we investigated their fate after exposure to cellular environment by several fluorescence spectroscopy techniques. First, we elucidated the degradation of four differently modified, dual-dye labeled short RNA oligonucleotides in HeLa cell extracts by fluorescence correlation spectroscopy, fluorescence cross-correlation spectroscopy and Förster resonance energy transfer. We observed that the integrity of the oligonucleotide sequence correlates with the extent of chemical modifications. Furthermore, the data showed that nucleolytic degradation can only be distinguished from unspecific effects like aggregation, association with cellular proteins, or intramolecular dynamics when considering multiple measurement and analysis approaches. We also investigated the localization and integrity of the four modified oligonucleotides in cultured HeLa cells using fluorescence lifetime imaging microscopy. No intracellular accumulation could be observed for unmodified oligonucleotides, while completely stabilized oligonucleotides showed strong accumulation within HeLa cells with no changes in fluorescence lifetime over 24 h. The integrity and accumulation of partly modified oligonucleotides was in accordance with their extent of modification. In highly fluorescent cells, the oligonucleotides were transported to the nucleus. The lifetime of the RNA in the cells could be explained by a balance between release of the oligonucleotides from endosomes, degradation by RNases and subsequent depletion from the cells.

Monitoring drug nanocarriers in human blood by near-infrared fluorescence correlation spectroscopy

“…With its sensitivity down to the level of single dye molecules, the method is suitable to study the stability, premature release, interaction with blood components or aggregation of NCs in blood. Although our focus was on monitoring of NCs loading stability, the method can be also applied to study the size or detect possible interactions of other exogenously introduced NIR labeled species, such as proteins, RNA, or DNA 48,49 .…”

A critical analysis of methods used to investigate the cellular uptake and subcellular localization of RNA therapeutics

“…Other, more advanced fluorescence-based techniques have been implemented to address some of the drawbacks of flow cytometry and fluorescence microscopy (Figure 3C ) ( 87 , 88 ). Fluorescence lifetime imaging microscopy (FLIM) can measure the total cellular uptake and stability of RNA therapeutics in cultured cells ( 52 , 89–93 ). The fluorescence lifetime of a fluorophore can be altered when it is in close proximity to another fluorophore or when it is in different chemical environments, such as packaged in a delivery vector, trapped in an endosome, or free in the cytosol ( 91 , 94 , 95 ).…”

“…More advanced fluorescence techniques are capable of better quantitation of the amount of dye-labeled molecule within the cell, and they can also better distinguish subcellular compartments. A key example is fluorescence correlation spectroscopy (FCS) ( 138 ), which has been applied to investigate the concentrations of RNA therapeutics in live cells (Figure 5A ) ( 52 , 53 , 91 , 93 ). In FCS, the diffusion of a dye-labeled molecule is tracked within a small (femtoliter) focal volume by measuring fluctuations in fluorescence intensity of molecules entering and exiting the focal volume.…”

“…The Schwille group used FCCS to analyze the incorporation of microinjected dye-labeled siRNAs into the RISC complex, visualized using an expressed Argonaute2-EGFP fusion in both the cytosol and nucleus ( 142 ). In another example, researchers in the Wagner and Lamb groups at the University of Munich used FCCS to assess the cytosolic degradation of dual-labeled oligonucleotides through simultaneous tracking of both fluorophores of a FRET pair ( 52 ).…”

Solid-phase supported design of carriers for therapeutic nucleic acid delivery

“…Nuclear import is a crucial size-dependent process, and presents the next important barrier for delivery of larger nucleic acids such as pDNA [ 12 ]. The nuclear pore complex (NPC) only allows the passage of small molecules such as oligonucleotides [ 176 , 205 , 206 ]. whereas polyplexes greater than ∼50 nm do not have this capacity.…”