Watermelon chemistry: silver nanoclusters for enzyme detection

Welcome to Plenty of Room! Today, I’m excited to share a paper that, while not brand new, is still fairly recent: and more importantly, incredibly interesting!

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Watermelon chemistry: silver nanoclusters for enzyme detection

I talk a lot about the nanoscale here, and nanoparticles are no new concept: tiny particles between 1 and 100 nm in size. I usually think of gold or silver nanoparticles, quite common in sensors, but there are others, like silica or iron, and my favorite, DNA origami, also belongs in there.

What I did not even know existed are nanoclusters. These are tinier than nanoparticles, ranging from just 1 to 10 nm, made up of only a handful of atoms. We’re literally talking about clusters composed of 5 to 100 atoms! And when it comes to metal nanoclusters, these miniature assemblies exhibit fascinating optical and electronic properties.

Today’s paper explore the biomedical potential of DNA-templated silver nanoclusters. Combining DNA with silver nanoclusters gives rise to unique fluorescent properties, dependent on the DNA sequence and structure. Fluorescence is everywhere in biology, and here, the focus is on creating a low-cost alternative to the popular (but expensive) FRET sensors used for detecting nuclease activity.

To do this, the team introduced Subak, a DNA-templated silver nanocluster probe, named after the Korean word for “watermelon” (up there for name of the year, in my book). Why watermelon? Well, just like a watermelon that’s green on the outside and turns red when cut open, Subak glows green (540 nm) when intact and shifts to bright red (630 nm) after a nuclease digests the DNA. Genius!

Unlike FRET-based sensors, Subak works through cluster transformation. Upon nuclease digestion, the structure of the silver nanocluster changes, causing the dramatic color shift. So, how does this cool (and cheap) tool actually get used?

The researchers actually created two probes, Subak-1 and Subak-2, for two different applications:

• DNAase I detection: Subak-1 was used to detect the activity of DNAase I, a DNA-cleaving enzyme. The green-to-red fluorescence shift confirmed DNase I activity, validating the reporter’s use in enzyme activity assays.

• CRISPR-Cas12a DETECTR Assays: DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) is a diagnostic tool that utilizes the Cas12a enzyme to recognize specific DNA sequences and cleave a reporter molecule. Traditionally this essay used double labeled FRET reporters, that here were changed for DNA templated silver nanoclusters. In the presence of a target DNA sequence, Cas12a is activated and cleaves the Subak-2 DNA strand, triggering the fluorescence shift. This allows for real-time detection of the CRISPR activity with a clear visual indicator.

Subak brings two major advantages over traditional FRET systems:

1. Cost: FRET reporters cost around $62 per nanomole, but Subak drops that down to just $1 per nanomole. That’s a huge deal for scaling up experiments or making molecular diagnostics more affordable.

2. Simplicity: Subak’s straightforward mechanism opens up possibilities for broader adoption, especially in resource-limited settings where cost and accessibility are key.

In the paper there is actually a lot more, including a detailed structural analysis of the silver nanoclusters before and after the transitions. So, if you are interested, just go here and enjoy the full paper!

Additional room:

• More precise DNA-vesicle sensors: If you can’t get enough of vesicles or sensors, this is the paper for you! The team here introduces a DNA origami-based biosensor for detecting lipid vesicles and delivering cargo using single-molecule FRET (smFRET). The sensor features a hydrophobic ssDNA that coils in a vesicle-free environment, maintaining high FRET efficiency. Upon binding to vesicles, the ssDNA stretches, reducing FRET efficiency to signal vesicle interaction. The ssDNA can also transfer cargo, allowing controlled release!

• Better composite DNA materials: High up on my reading list is this review, which explores the growing use of DNA as a building block for new materials. In particular, it focuses on DNA-based molecular complexes and composites that have been developed, combining the physical and chemical properties of inorganic or organic molecules with DNA's unique functions. These materials show great potential in materials science, nanotechnology, and biomedical engineering. However, challenges like high production costs, biological stability, and potential immunogenicity pose obstacles to their broader application. Let’s see if I can get some new ideas!

• Growing DNA origami filaments: This study shows how you can control the assembly and disassembly of DNA origami filaments (DOF) using an autocatalytic DNA reaction network (DRN). The filaments grow through fuel release from the DRN, while toehold-mediated strand displacement triggers disassembly. This approach allow for precise control over DNA nanomaterials, enabling programmable responses to external stimuli!

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