Advanced Molecular Imaging with Raman Cluster Tags for Precise Biological Insight

Introduction

In the field of medical diagnostics and biotechnology research, the ability to visualize and track biological molecules at the cellular level is critical for understanding disease mechanisms and developing new therapies. Conventional imaging methods, while effective, often face challenges in resolution, sensitivity, and specificity. Our patented Raman cluster tagged molecules offer a groundbreaking solution for biological imaging, providing a highly sensitive and precise approach to visualize molecular interactions within complex biological environments.

Limitations of Traditional Imaging Techniques

Traditional imaging methods, such as fluorescence or magnetic resonance imaging (MRI), are widely used in both research and clinical settings, but they come with certain limitations. Fluorescence imaging, for example, can suffer from photobleaching, low resolution, and interference from background signals, which affects its sensitivity and accuracy. MRI, while useful for anatomical imaging, may lack the molecular specificity needed for detecting subtle biological changes at the cellular level.

In the context of molecular biology, researchers need a method that can accurately track biological processes and molecular interactions in real-time, without interference from noise or degradation. This need is particularly pressing in areas such as cancer research, drug development, and genomics, where detecting changes at the molecular level is key to advancing science and improving patient outcomes.

A New Dimension of Precision with Raman Cluster Tags

Our patented Raman cluster tagged molecules introduce a new standard in molecular imaging, offering unparalleled sensitivity and specificity for detecting and visualizing target molecules. These cluster-tagged molecules take advantage of Raman scattering, a phenomenon that provides highly specific vibrational information about molecules. Unlike traditional imaging techniques, Raman imaging is not affected by photobleaching, and the signal remains strong and stable over time.

The use of these tagged molecules enables researchers and clinicians to track molecular interactions with incredible accuracy, even within complex biological systems. This opens up a wide range of applications, from early cancer detection to monitoring drug efficacy in real-time. Moreover, the ability to detect multiple signals simultaneously makes this technology ideal for multiplexing applications, where various biomarkers need to be monitored in parallel.

Advantages of Raman Cluster Tagged Molecules

Enhanced Sensitivity and Specificity: Raman scattering provides highly detailed molecular information, allowing for accurate detection of target molecules.
Stable and Durable: Unlike fluorescence imaging, Raman tags do not suffer from photobleaching, ensuring stable, long-lasting signals.
Broad Applications: From cancer diagnostics to drug discovery, this technology can be applied across various fields in medical research and biotechnology.
Multiplexing Capability: The ability to detect multiple molecules simultaneously makes it a powerful tool for complex biological studies.

A Game-Changer in Molecular Imaging

Licensing this Raman cluster tagged molecule technology offers an opportunity to lead in advanced molecular imaging, providing researchers and clinicians with a tool that expands the boundaries of what is possible in biological research and diagnostics. With its superior sensitivity, durability, and versatility, this technology represents a critical advancement in the precision and accuracy of biological imaging.

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Abstract
Claims

This invention provides nucleoside polyphosphate analogs each of which comprises a tag comprising a plurality of Raman-scattering moieties; compounds comprising said nucleoside polyphosphate analogs; and methods for determining the sequence of a single-stranded DNA or RNA using said nucleoside polyphosphate analogs. This invention also provides methods for detecting the interaction of a plurality of predetermined compounds, at least one of which having attached thereto a tag comprising a plurality of Raman-scattering moieties.

What is claimed is:

1. A nucleoside polyphosphate analog having the structure:

wherein B is a base and is adenine, guanine, cytosine, uracil, thymine, or a derivative thereof, wherein R₁is OH, wherein R₂is OH or H, wherein n is 1, 2, 3, or 4, wherein X is a linker, and wherein the tag comprises a plurality of identical Raman-scattering moieties.

2. The nucleoside polyphosphate analog of claim 1, wherein each of the plurality of Raman-scattering moieties has a Raman spectroscopy peak with wavenumber from 2000 cm⁻¹to 2300 cm⁻¹.

3. The nucleoside polyphosphate analog of claim 1, wherein each of the plurality of Raman-scattering moieties is selected from the group consisting of —N═N═N, —C≡N, —C≡CH, and —C≡C—CH₃.

4. The nucleoside polyphosphate analog of claim 1, wherein the tag further comprises at least one Raman-scattering moiety that is different from the identical Raman-scattering moieties of the plurality of identical Raman-scattering moieties.

5. The nucleoside polyphosphate analog of claim 1, wherein the tag comprises 3 identical Raman-scattering moieties.

6. The nucleoside polyphosphate analog of claim 1, wherein the plurality of Raman-scattering moieties forms a linear tag.

7. The nucleoside polyphosphate analog of claim 1, wherein the plurality of Raman-scattering moieties forms a non-linear tag.

8. The nucleoside polyphosphate analog of claim 7, wherein the non-linear tag is a dendrimer tag.

9. The nucleoside polyphosphate analog of claim 2, wherein the tag has a Raman spectroscopy peak with wavenumber from 2125 cm⁻¹to 2260 cm⁻¹.

10. The nucleoside polyphosphate analog of claim 1, wherein the linker is selected from the group consisting of O, NH, S, CH₂, amino acids, peptides, proteins, carbohydrates, polyethylene glycols of different length and molecular weights, aliphatic acids, aromatic acids, alcohols or thiol groups (substituted or unsubstituted), cyano groups, nitro groups, alkyl groups, alkenyl groups, alkynyl groups, and azido groups.

11. A composition comprising four deoxyribonucleoside polyphosphate (dNPP) analogs, each dNPP analog having the structure:

wherein B is a base and is adenine, guanine, cytosine, thymine, or a derivative thereof, wherein R₁is OH, wherein R₂is H, wherein n is 1, 2, 3, or 4, wherein X is a linker, wherein the tag comprises a plurality of identical Raman-scattering moieties, wherein (i) the Raman spectroscopy peak of the tag on each dNPP analog is distinguishable from the Raman spectroscopy peak of the tag on each of the remaining three dNPP analogs, and (ii) each dNPP analog comprises a base which is different from the base of each of the remaining three dNPP analogs.

12. A composition comprising four ribonucleoside polyphosphate (rNPP) analogs, each rNPP analog having the structure:

wherein B is a base and is adenine, guanine, cytosine, uracil, or a derivative thereof, wherein R₁is OH, wherein R₂is OH, wherein n is 1, 2, 3, or 4, wherein X is a linker, wherein the tag comprises a plurality of identical Raman-scattering moieties, wherein (i) the Raman spectroscopy peak of the tag on each rNPP analog is distinguishable from the Raman spectroscopy peak of the tag on each of the remaining three rNPP analogs, and (ii) each rNPP analog comprises a base which is different from the base of each of the remaining three rNPP analogs.

13. A method for determining the sequence of a single-stranded DNA comprising:

(a) contacting the single-stranded DNA having a primer hybridized to a portion thereof with a DNA polymerase and four deoxyribonucleoside polyphosphate (dNPP) analogs under conditions permitting the DNA polymerase to catalyze incorporation onto the primer of a dNPP analog complementary to a nucleotide residue of the single-stranded DNA which is immediately 5′ to a nucleotide residue of the single-stranded DNA hybridized to the 3′ terminal nucleotide residue of the primer, so as to form a DNA extension product, wherein each dNPP analog has the structure:

wherein (i) the Raman spectroscopy peak of the tag on each dNPP analog is distinguishable from the Raman spectroscopy peak of the tag on each of the remaining three dNPP analogs, and (ii) each dNPP analog comprises a base which is different from the base of each of the remaining three dNPP analogs, and

wherein the incorporation of the dNPP analog releases a tagged polyphosphate;

(b) determining the wavenumber of the Raman spectroscopy peak of the tagged polyphosphate released in step (a), so as to thereby determine the identity of the incorporated dNPP analog and the identity of the complementary nucleotide residue in the single-stranded DNA; and

(c) iteratively performing steps (a) and (b) for each nucleotide residue of the single-stranded DNA to be sequenced so as to thereby determine the sequence of the single-stranded DNA.

14. A method for determining the sequence of a single-stranded RNA comprising:

(a) contacting the single-stranded RNA having a primer hybridized to a portion thereof with an RNA polymerase and four ribonucleoside polyphosphate (rNPP) analogs under conditions permitting the RNA polymerase to catalyze incorporation onto the primer of an rNPP analog complementary to a nucleotide residue of the single-stranded RNA which is immediately 5′ to a nucleotide residue of the single-stranded RNA hybridized to the 3′ terminal nucleotide residue of the primer, so as to form an RNA extension product, wherein each rNPP analog has the structure:

wherein (i) the Raman spectroscopy peak of the tag on each rNPP analog is distinguishable from the Raman spectroscopy peak of the tag on each of the remaining three rNPP analogs, and (ii) each rNPP analog comprises a base which is different from the base of each of the remaining three rNPP analogs, and

wherein the incorporation of the rNPP analog releases a tagged polyphosphate;

(b) determining the wavenumber of the Raman spectroscopy peak of the tagged polyphosphate released in step (a), so as to thereby determine the identity of the incorporated rNPP analog and the identity of the complementary nucleotide residue in the single-stranded RNA; and

(c) iteratively performing steps (a) and (b) for each nucleotide residue of the single-stranded RNA to be sequenced so as to thereby determine the sequence of the single-stranded RNA.

15. The method of claim 13, wherein the polymerase is immobilized on a solid surface.

16. The method of claim 13, wherein the Raman spectroscopy peak is determined using surface-enhanced Raman spectroscopy (SERS).

17. The method of claim 16, wherein the polymerase is immobilized on a SERS substrate.

18. The method of claim 17, wherein the polymerase is immobilized such that only the tagged polyphosphate released in step (a) is within the detection range of the SERS detector.

19. The method of claim 18, wherein the polymerase is immobilized such that the tagged polyphosphate released in step (a) is within a cavity on the SERS substrate.

20. The method of claim 13, wherein the linker is selected from the group consisting of O, NH, S, CH₂, amino acids, peptides, proteins, carbohydrates, polyethylene glycols of different length and molecular weights, aliphatic acids, aromatic acids, alcohols or thiol groups (substituted or unsubstituted), cyano groups, nitro groups, alkyl groups, alkenyl groups, alkynyl groups, and azido groups.

Title

Raman cluster tagged molecules for biological imaging

Inventor(s)

Jingyue Ju, Shiv Kumar, Mirkó PALLA, James J. Russo

Assignee(s)

Columbia University in the City of New York

Patent #

10648026

Patent Date

May 12, 2020