In 1900, German physician Paul Ehrlich came up with the idea of a “magic bullet.” The concept was to inject a patient with smart particles capable of finding, recognizing, and treating disease.

Medical science has pursued the magic bullet ever since.

Russian researchers from the Moscow Institute of Physics and Technology and Prokhorov General Physics Institute, RAS, have made headway toward that goal. Led by MIPT’s Maxim Nikitin, the team has presented a smart material with unique properties, which holds promise for express DNA analysis and next-generation drugs against cancer and other serious diseases[1].

A Major Bottleneck

Delivering medications to the cells affected by a disease is a significant bottleneck in diagnostics and therapy. The drugs should ideally reach the pathogenic cells only, without doing any harm to the healthy ones.

A range of marker compounds that give away cancer cells exist. Among these molecules, found on the surface of the affected cells or in their microenvironment, are waste products and those sent to other cells as signals.

Modern drugs rely on one such marker to identify diseased cells. However, it is usually the case that healthy cells carry the same tags, albeit in smaller quantities, meaning existing targeted drug delivery systems are not perfect.

Better Deliveries

We need smart materials that are capable of analyzing multiple environment parameters at once, seeking out the target with higher precision.

“The conventionally used methods for drug delivery are like sending a letter with the city and street written on the envelope, but without the house and apartment numbers. We need to be able to analyze more parameters to ensure effective delivery,“

Maxim Nikitin, the principal investigator and the head of MIPT’s Nanobiotechnology Lab, commented.

Previously, Nikitin and co-authors developed nano- and microparticles capable of conducting complex logic computations via biochemical reactions. In a 2014 paper[2], the researchers reported that their autonomous nanocomputers could analyze many parameters of a target and were therefore much better at its identification.

The past few years have seen many advances in biocomputing materials. By 2018, hundreds upon hundreds of papers were published on the subject.

Chemical Reviews, the field’s most reputable journal, published a review of contemporary nanorobotics and biocomputing. The paper, with the subtitle “Dawn of Theranostic Nanorobots,” was authored by researchers from MIPT’s Nanobiotechnology Lab and the Biophotonics Lab of Prokhorov General Physics Institute of the Russian Academy of Sciences (RAS).

DNA Supersensitivity

Despite the efforts of numerous research teams around the world trying to expand the functionality of biocomputing materials, they are still not sensitive enough to disease markers, rendering practical applications impossible.

This recent paper marks a breakthrough in this field and features a unique smart material characterized by supersensitivity to DNA signals. It is several orders of magnitude more sensitive than the closest competitor.

Moreover, the new material exhibits higher sensitivity than that of the vast majority of currently available express DNA assays.

The researchers achieved that remarkable result after they discovered that DNA molecules exhibit unusual behavior on the surface of nanoparticles.

Researchers pinned one end of a single-stranded DNA molecule to a nanoparticle. Importantly, the molecule had no hairpins — that is, double-stranded segments where part of the chain sticks to itself.

The team outfitted the other end of the DNA chain with a small molecular receptor. Contrary to expectations, the receptor did not bind its target.

After ruling out a mistake, the scientists hypothesized that single-stranded DNA might stick to the nanoparticle and coil up, hiding the receptor beneath it, on the particle’s surface.

A Chameleon Tongue

The hypothesis proved right when the team added complementary single strands of DNA to their particle. The receptor instantly became active, binding its target.

Bonds between the complementary nucleotides caused the two DNA strands to form a rigid double helix. Like a chameleon’s tongue, the strand uncoiled, exposing the receptor for target binding.

[caption id="attachment_102505” align="aligncenter” width="700”]Adding a complementary DNA strand activates the receptors on the nanoparticle surface Adding a complementary DNA strand activates the receptors on the nanoparticle surface.
Credit: Vladimir Cherkasov et al.[/caption]

Such uncoiling of the DNA strand resembles that of a molecular beacon - a single-stranded DNA whose one end forms a duplex with the opposite end, folding up the structure.

A complementary strand of DNA can unfold the beacon. However, there is a significant and useful distinction.

“Unlike molecular beacons, the discovered phenomenon enables tuning the force of DNA curling on the nanoparticle separately from the straightening force of input DNA. This leads to dramatically better sensitivity to the input,“

noted the study’s first author Vladimir Cherkasov, a leading researcher at the Nanobiotechnology Lab, MIPT.

Practical Applications

In their paper, the researchers demonstrate agents capable of detecting DNA concentrations as low as 30 femtomoles (30 billionths of a millionth of a mole) per liter, without DNA or signal amplification.

“We showed the sensitivity to be so high with a quite simple lateral flow assay, which is widely used in pregnancy tests. Unlike the existing DNA assays, such tests can be performed outside a clean laboratory setting and require no advanced equipment. This makes the technology well-suited to rapid infectious disease screening, food testing kits for home use, and similar things,“

The study’s co-author Elizaveta Mochalova added.

The authors of the paper have also shown the technology to apply to the design of smart nano-agents that would recognize cancer cells based on the concentration of small DNA in their microenvironment.

Not long ago, small nucleic acids were thought to be just meaningless debris resulting from the recycling of larger functional molecules. However, small RNAs turned out to be critical regulators of many processes in living cells. Biologists are currently identifying disease markers among these RNAs.

“Interestingly, the smaller the length of the nucleic acid to be detected, the more competitive our technology becomes. We can fabricate ultrasensitive agents controlled by well-studied small RNAs that are 17 to 25 bases long. However, if we take sequences that are less than ten nucleotides long, there are simply no technologies with comparable sensitivity. What’s even more exciting is that our method enables probing the microenvironment of cells to determine whether shorter small RNAs are useful disease markers rather than the meaningless compounds they are commonly held to be due to the difficulties in their detection,“

Nikitin commented.

The newly developed technology offers prospects for genomics, both in terms of point-of-care DNA assays and for developing next-generation therapeutic nanomaterials. The recent years have seen immense breakthroughs in genome research and editing. Still, the new technology could solve the problem that remains relevant: delivering drugs only to the cells with a particular microenvironment genetic profile.

[1] Vladimir R. Cherkasov, Elizaveta N. Mochalova, Andrey V. Babenyshev, Alexandra V. Vasilyeva, Petr I. Nikitin, Maxim P. Nikitin. Nanoparticle Beacons: Supersensitive Smart Materials with On/Off-Switchable Affinity to Biomedical Targets. ACS Nano 2020, 14, 2, 1792-1803

[2] Nikitin, M., Shipunova, V., Deyev, S. et al. Biocomputing based on particle disassembly. Nature Nanotech 9, 716–722 (2014). https://doi.org/10.1038/nnano.2014.156

Top image: Vladimir Cherkasov et al./ACS Nano


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