Squaramide-Linked Oligonucleotide Synthesis via Squarate Conjugation
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Solution Overview
Problem
The standard phosphoramidite solid phase synthesis method for preparing long oligonucleotides faces challenges in coupling efficiencies and purification, especially as lengths exceed 70 bases, making it difficult to synthesize and purify oligonucleotides beyond 100-150 mers at mmole scales, and enzymatic ligations are required for longer sequences.
Innovation Solution
The method involves activating a terminal of one oligonucleotide using a squarate reagent to form a squaramide linkage with another oligonucleotide via a splint oligonucleotide, allowing for the conjugation of oligonucleotides to produce squaramide-linked oligonucleotides, enabling the synthesis of longer sequences by forming internucleoside squaramide linkages.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If standard phosphoramidite solid phase synthesis method is used to prepare long oligonucleotides, then synthesis can be performed at 1-10 μmole scale, but purification of Full Length Product from truncated species becomes increasingly difficult as length grows beyond 70 bases
Solution Approach 1:
The patent divides the long oligonucleotide synthesis into multiple segments or blocks. Instead of synthesizing the entire long sequence in one continuous phosphoramidite coupling process, the oligonucleotide is synthesized in smaller blocks that are then ligated together using squarate chemistry. This segmentation allows each block to be synthesized and purified separately at manageable lengths, avoiding the purification difficulties associated with synthesizing entire long sequences in one step.
Solution Approach 2:
The patent introduces squarate linkages as intermediary chemical structures that facilitate the joining of oligonucleotide blocks. The squarate reagent acts as a mediator that reacts with terminal amines of separate oligonucleotide blocks to form stable squaramide bonds, enabling efficient ligation of pre-synthesized blocks into longer sequences without requiring enzymatic intervention.
2Length of stationary object
If oligonucleotide length exceeds 70 bases, then longer sequences can be achieved, but practical synthesis and purification limits are reached at approximately 70 bases at mmole scales and 100-150 mers at 1 umole scale
Solution Approach 1:
The patent applies segmentation by dividing the long oligonucleotide into multiple shorter blocks that can be independently synthesized using standard phosphoramidite chemistry at manageable lengths (within the 70-150 base feasible range). These blocks are then joined through squarate-mediated ligation to achieve final lengths exceeding 300 bases, thereby overcoming the synthesis feasibility limits of conventional single-step methods.
Solution Approach 2:
The patent employs preliminary action by pre-synthesizing and purifying individual oligonucleotide blocks to a high degree of purity before ligation. This preliminary synthesis and purification of blocks at optimal lengths ensures that when they are ligated together, the final long oligonucleotide achieves both extended length and high overall purity, bypassing the limitations of direct long-sequence synthesis.
3Productivity
If conventional methods are used for oligonucleotide conjugation, then standard phosphodiester linkages are formed, but coupling efficiencies decrease and purification becomes increasingly difficult as length grows
Solution Approach 1:
The patent changes the chemical parameters of the linkage chemistry by replacing standard phosphodiester bond formation with squarate-based coupling chemistry. The squarate reagent reacts with terminal amines to form squaramide linkages, which have different chemical properties and reaction kinetics compared to phosphodiester bonds. This parameter change in chemistry enables efficient coupling of oligonucleotide blocks with high yields and simplified purification protocols, even for very long final sequences.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach facilitates the synthesis of longer oligonucleotides by improving coupling efficiencies and simplifying purification, allowing for the production of oligonucleotides with multiple squaramide linkages, overcoming the limitations of traditional methods in length and scale.
Implementation Method 1
activating a terminal of first oligonucleotide using a squarate reagent to produce an activated first oligonucleotide; and binding the first oligonucleotide and a second oligonucleotide to a splint oligonucleotide; to conjugate the activated first oligonucleotide with a terminal of the second oligonucleotide via a squaramide linkage
Data Source
AI summary
Methods of conjugating oligonucleotides are provided. The methods may include: activating a terminal of first oligonucleotide using a squarate reagent to produce an activated first oligonucleotide; and binding the first oligonucleotide and a second oligonucleotide to a splint oligonucleotide; to conjugate the activated first oligonucleotide with a terminal of the second oligonucleotide via a squaramide linkage to produce a squaramide-linked oligonucleotide. Also provided are oligonucleotides that include a squaramide internucleoside linkage. Compositions are provided that include a first oligonucleotide including 300 or more nucleosides and at least one squaramide internucleoside linkage; and a second complementary oligonucleotide not including a squaramide linkage. Kits and compositions for practicing the subject methods are also provided.