Nucleic acid structures

Nucleic acids (DNA and RNA) are fundamental components of all forms of life. They function to store, transmit, and express genetic information. In these processes, DNA and RNA structures play central roles. Taking advantage of our optical tweezers, we have studied a variety of DNA and RNA structures to reveal their implications for life activities.

DNA forms not only the canonical duplex structure but also non-canonical structures like G-Quadruplex, i-Motif, cruciform, hairpin, etc. All these structures have functional significance in a cell. Using magneto-optical tweezers we have discovered that DNA G-quadruplex, i-Motif, and cruciform can modulate transcription, which is the process of producing RNA from DNA, by adjusting their properties according the mechanical force (Zhongbo Yu, JACS 2009; Soma Dhakal, JACS 2010) or the torque experienced (Ref: Sangeetha Selvam, JACS, 2014; Shankar Mandal, ChemPhysChem, 2018). The G-quadruplexes are particularly present in promoter region of DNA and regulates gene-expressions. We have studied those G-Quadruplexes from mechanochemical perspective (Ref: Soma Dhakal, Biophysical Journal, 2012; Sangeetha Selvam, JACS, 2014; Chiran Ghimire, JACS, 2014). Nucleic acid strands also form hairpin structures. Accordingly, we have investigated the properties of B-DNA using hairpin as a model (Ref: Sagun Jonchhe, JACS, 2020). This study was performed inside nanocavity to mimic the nanoconfinement in cellular machineries such as transcription complexes.

Aptamers are the oligonucleotides that bind specifically to a target molecule. We have used those aptamers to recognize targets by the measurement of interaction force between the target and aptamer probes (Ref: Philip, AnalChem, 2012).

Like DNA, RNA also forms secondary structures. Our lab has studied the long noncoding telomeric RNA (TERRA) and discovered the formation of intramolecular G-Quadruplexes with increased mechanical stability and partially folded structures in human TERRA (Ref: Philip, ChemBioChem, 2013). We also have studied the molecular interactions between binding ligands and RNA GQs (Philip, Angewandte), as well as those between DNA and RNA GQs at the same location in the template (Ref: Prakash, NAR, 2014).

In summary, we have investigated the nucleic acid secondary structures at single-molecule level to reveal their properties and significance from the mechanochemical perspective.

Application using nucleic acids

  • Single molecule mechanochemical sensing
  • A biosensor is an analytical device comprising biological components for the detection of a chemical substance. Biosensors are mainly composed of three elements: probes for analyte recognizing, amplification components, and signal reporters. Usually, high sensitivity biosensors require multiple amplification components, which creates long transmission time between the probe and the reporter. This long transmission window limits the real-time response (ChemPhysChem, 2015, 16:1829-37). To overcome this issue and simultaneously keep the high sensitivity, we have designed a mechanophore where the single-molecule recognition probe and signal reporter are integrated in a single DNA template. Exploiting the stochastic behavior (Deepak JACS 2011) of a single-molecule mechanophore in a microfluidic platform coupled with optical tweezers, we have reached ultimate single-molecule sensitivity with real-time detection. Moreover, multiplex tasking has also been achieved by incorporation of different mechanophores in the same single-molecule template (Shankar Mandal, Anal. Chem. 2019; Sagun, Nature Materials, 2020).

  • Bio-nanotechnology
  • Mechanical property of DNA origami self-assembly
  • DNA nanotechnology is a relatively new field which has potential application in various fields including nanomaterials, drug delivery and biosensing. The field exploits specificity of complementary DNA base pairing to design and synthesize various 2D and 3D DNA structures at nanometer level precision. DNA origami is a technique in the field that involves folding of single stranded DNA scaffold into desired nanostructure by DNA staples that form Holliday junctions with the scaffold. Limited by current methodologies, however, mechanical properties of DNA origami structures have not been adequately characterized, which hinders further applications of these materials. To this part, we intend to study mechanochemical properties of different nanoassemblies which will open further avenues for better design of the nanoassemblies and its applications. We have studied the mechanical properties of various DNA nanoassemblies: nanotubes, nano-tiles, and nanopyramid using optical tweezers. We found that the mechanical property of the nanoassembly is governed by Holliday junctions along a stress direction. The mechanical properties observed here certainly provide insights for designing highly stable DNA nanostructures.

  • Nanoconfinement
  • Various cellular processes like replication and transcription are carried out in nanocavities inside cellular machineries. The cellular processes are often mediated via biomolecules in their folded or unfolded/misfolded forms. Previous researches suggest that these nanocavities have pronounced effect on the stability of biomolecules such as DNA, RNA, and proteins. We are interested in investigating the stability of different biomolecular structures inside nanoconfinement (Nature Nanotechnology 2017, PNAS 2018, JACS 2020). Placing non-B DNA and B DNA (i.e. natural duplex DNA) structures inside the DNA origami nanocages, we discovered enhanced stability of non-B DNA and reduced stability of B DNA which are mediated via reduced water activity inside the DNA nanocages. These promising results suggest an alternative pathway to populate non-B DNA over B DNA structures in nanoconfinement, which offer a unique way to modulate cellular processes.

    Molecular interactions

  • Synthetic host-guest interactions
  • Synthetic host-guest chemistry has become a versatile and dynamic field for drug delivery, catalysis, sensing, and protein targeting. It includes macrocyclic host molecules (like Crown ethers, Cryptands, Cyclodextrins, Cucurbiturils, Calixarene, Calixpyrroles, etc.) with have an empty pocket and the guest molecules (gaseous molecules, metal ions, drugs, organic compounds, biomolecules, etc.) which have a specific size and functional groups. It is based on the recognition between the host and the guest with high affinity and specificity due to proper size fitting and polarity matching (like dissolves like). Non-covalent interactions like H-bonding, Van der Waals forces, electrostatic or hydrophobic interaction are important for the host-guest chemistry. However, the extent of non-covalent force inside the host-guest complex is still ambiguous.

    We have developed a single-molecule platform to test the kinetics and mechanical stability of the host-guest interaction by conjugating the host and guest into DNA fragments via click chemistry. In our lab, we have successfully studied the mechanical stability of Cucurbit[7]uril-adamantane and β-cyclodextrin-adamantane complexes by means of optical tweezers. Using this method, mechanical stability of various host-guest pairs can be further exploited in various biomedical, biosensing, and materials fields (Pandey, JACS, 2019).

  • Ligand binding to DNA structures (polyamide, telomestatin and derivatives, PDS)
  • Many factors contribute to the uncontrolled growth of cancer cells. One of them is the improper functioning of telomerase enzyme which is involved in maintaining the length of chromosomes during cell division. Over past decades, it is known that at the end of chromosomes, DNA sequences comprise of a repeat of non-canonical forms (G-quadruplex) along with the predominant double-helices. During the elongation of this DNA region, these structures unfold and allow unrestricted access of the telomerase. However, when these structures are stabilized using ligands, the elongation can be halted. Based on that principle, to restrict the reverse transcription of the telomerase enzyme, we have conjugated G-quadruplex binding ligand such as pyridostatin with duplex DNA recognition elements such as polyamides. This dual targeting maintains both high binding affinity and selective recognition between G-quadruplex forming region and the conjugated ligands (Shankar, NAR 2019). In another approach, we have also studied the mechanical property of individual human telomeric G-quadruplexes bound to telomestatin derivatives (Chiran Ghimire, JACS; Sagun, Angewandte 2019). These studies aim to develop drugs that have high affinity and specificity for oncogenes.

    Instrumentation and methodology development

    Optical tweezers provide the possibility to manipulate individual particles or droplets in micrometer size, or to input heat into a small volume. Exploiting those advantages, we established several methodologies to study different molecular phenomena. By moving one of the optically trapped beads quickly, we can change the force applied on a single-molecule template tethered between two optically trapped beads instantly to achieve force jump, which has been used to study the kinetics of G-Quadruplex folding and unfolding (Deepak, Nat. Chem., 2011). By moving two optically trapped beads across the boundary between two buffers, we could achieve concentration jump (Sagun, Anal. Chem., 2018), which was primarily used to investigate single-molecule kinetics. Likewise, by focusing a low intensity laser on a black microparticle, we achieved temperature jump in an extremely small volume (zeptoliter) without photo damaging (Deepak, Angew. Chem., 2014), offering a platform for single-molecule temperature control. Another methodology development was carried out by manipulating droplets and monitoring the optical signal or combining other characterization methods. Here we successfully characterized individual micrometer-sized droplets in situ and studied droplet fusion (Mao, Sensors and Actuators B, 2008; Chiran, Langmuir, 2014). In yet another direction, we invented a brand-new magneto-optical tweezers in which a magnetic field was used to achieve torsional control on single molecules tethered between optically-trapped beads for their mechanical investigations (Sangeetha, JACS, 2014).